Administration of kynurenine depleting enzymes for tumor therapy

ABSTRACT

Methods and compositions related to the use of a protein with kynureninase activity are described. For example, in certain aspects there may be disclosed a modified kynureninase capable of degrading kynurenine. Furthermore, certain aspects of the invention provide compositions and methods for the treatment of cancer with kynurenine depletion using the disclosed proteins or nucleic acids.

The present application is a divisional of U.S. application Ser. No.15/351,060, filed Nov. 14, 2016, which is a divisional of U.S.application Ser. No. 14/473,040, filed Aug. 29, 2014, now U.S. Pat. No.9,808,486, which claims the priority benefit of U.S. provisionalapplication Nos. 61/872,132, filed Aug. 30, 2013 and 61/986,366, filedApr. 30, 2014, the entire contents of each of which are incorporatedherein by reference.

The invention was made with government support under Grant No. R01CA154754 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The invention generally relates to compositions and methods for thetreatment of cancer with enzymes that deplete L-kynurenine orL-3-hydroxykynurenine. More particularly, it concerns the engineering,pharmacological optimization and use of bacterial and mammalian enzymeswith L-kynurenine degrading activity suitable for human therapy.

2. Description of Related Art

Overexpression of indolamine-2,3-dioxygenase isoforms (IDO1 or IDO2) bycancer cells or reprogramming of cancer infiltrating leukocytes toexpress either of these enzymes has been shown to have a profound effecton attenuating adaptive immune responses to cancer. IDO1 and IDO2 aswell as the enzyme tryptophan 2,3-dioxygenase (TDO), whose expression bystromal cells may be induced by some tumors, catalyze the rate limitingstep in tryptophan (Trp) catabolism to L-kynurenine (KYN) (Godin-Ethieret al., 2011). Tumors exchange a cytosolic KYN molecule for anextracellular Trp molecule using a LAT1-like amino acid exchanger (Kaperet al., 2007), which has the dual effect on immune cells of locallyelevating levels of KYN while locally depleting Trp levels. Neighboringimmune cells internalize KYN, where it is an activating ligand for thearyl hydrocarbon receptor (AHR) resulting in the expression of numerouscytokines and chemokines that lead to tumor tolerance through immunecell differentiation and/or induction of apoptosis (Della Chiesa et al.,2006; Opitz et al., 2011; Song et al., 2011). Additionally, otherKYN-related compounds formed from kynurenine, most notably kynurenicacid also exert an immunosuppressive effect by serving as agonists ofthe orphan GPCR GPCR35. Inhibition of KYN formation (and thus inhibitionof the formation of KYN metabolism byproducts, including kynurenic acid,3-hydroxyl kynurenine and quinolinic acid, via the inhibition of IDO1 orTDO has received a significant amount of attention as a cancer target(Chen and Guillemin, 2009; Rutella et al., 2009; Prendergast, 2011).Substrate analog inhibitors, such as 1-DL-methyltryptophan, for IDO1have been developed and have shown initial promise in overcoming cancerinduced tumor tolerance thus restoring the ability of the native immunesystem to fight tumors (Lob et al., 2009). However, KYN is also producedby tryptophan 2,3-dioxygenase (TDO), which is also frequently expressedin tumors and this enzyme is not inhibited by 1-DL-methyltryptophan(Pilotte et al., 2012). There are also additional concerns with theD-isomer of 1-DL-methyltryptophan (1-D-MT) currently in phase 1 and 2clinical trials. Paradoxically, 1-D-MT can upregulate IDO1 expression,actually increasing KYN levels and inducing immunosuppression in certaincancers (Opitz et al., 2011).

Controlling tumor production of KYN is the focus of much research andhas the potential to treat, among others, cancers such as breast cancer,ovarian, glioblastoma, and pancreatic carcinoma. KYN is known tosuppresses proliferation as well as induce apoptosis in T cells and NKcells (Opitz et al., 2011; Mandi and Vacsei, 2012) enabling tumors toevade detection and destruction by a patient's immune system. KYN is apotent ligand of the aryl hydrocarbon receptor (AHR) whose activation inT cells induces differentiation into CD25+FoxP3+ T regulatory cells(Tregs) (Mezrich et al., 2010). KYN has also been shown to preventcytokine mediated up-regulation of specific receptors (NKp46 and NKG2D)required for NK mediated cell killing tumor cell lines (Della Chiesa etal., 2006), an action that is also likely mediated by its agonisticeffect on AHR (Shin et al., 2013). There is also clinical evidencelinking elevated serum KYN levels and decreased survival in multipletypes of cancer. In healthy patients, KYN levels in serum are in therange of 0.5 to 1 μM. In patients with cancer types that produce KYN,such as diffuse large B-cell lymphoma, serum KYN levels were measured tobe as much as 10 times higher (Yoshikawa et al., 2010; de Jong et al.,2011; Yao et al., 2011) and were prognostic for survival among lymphomapatients receiving the same treatment regimen; those with serum levelsbelow 1.5 μM exhibited a 3 year survival rate of 89%, compared to only58% survival for those with KYN levels above 1.5 μM. This difference insurvival was attributed to the immune suppressing effects of KYN(Yoshikawa et al., 2010). The use of small molecule IDO inhibitors, suchas 1-D-MT, has demonstrated the utility of controlling KYN levels inrestoring immune function, but the off target effects of IDO1up-regulation by 1-D-MT and lack of inhibition for TDO and the IDO1isoform are of concern.

The present invention discloses the use enzymes for the specificdepletion of KYN and its metabolites in tumors and/or in the blood. KYNdepleting enzymes are used to lower KYN concentrations for the treatmentof tumors expressing IDO1, IDO2, or TDO thus preventing tumor-mediatedtolerogenic effects and instead mediating tumor-ablatingpro-inflammatory responses. Notably, the use of enzymes for thedepletion of KYN and KYN metabolic byproducts circumvents the problemsassociated with small molecule inhibitors of IDO isoforms and TDOdiscussed above and further completely circumvents off target effectsthat are very commonly accompany small molecule drugs and lead tounpredicted toxicities and side effects.

SUMMARY OF THE INVENTION

Aspects of the present invention overcome a major deficiency in the artby providing enzymes that comprise bacterial and mammalian polypeptidesequences capable of degrading L-kynurenine and 3-hydroxy-L-kynurenineand displaying favorable pharmacokinetics in serum as desired for cancertherapy. In some aspects, the kynureninase enzyme may have greatercatalytic activity towards kynurenine than 3′ OH-kynurenine. Akynureninase from a bacterial species may be used. Such an enzyme mayhave an amino acid sequence selected from the group consisting of SEQ IDNOs: 7 and 13-52 or a modified version thereof. In particular, thetherapeutic may be derived from the Pseudomonas fluorescens enzyme,kynureninase (Pf-KYNU). Alternatively, a kynureninase from Saccharomycescerevisiae or Neurospora crassa may be used. The therapeutic may bederived from the Mucilaginibacter paludis kynureninase enzyme. Further,to prevent adverse effects due to the immunogenicity of heterologouskynureninases, the Homo sapiens enzyme or other primate kynureninasesdisplaying >95% sequence identity to the human enzyme may be used. Forexample, a novel enzyme may have an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 7-9.

In other aspects, there may be a polypeptide comprising either a nativeor modified human or primate kynureninase capable of degrading KYN andhaving activity towards the degradation of 3-hydroxykynurenine orkynurenic acid. In some embodiments, the polypeptide may be capable ofdegrading KYN under physiological conditions. For example, thepolypeptide may have a catalytic efficiency for KYN (k_(cat)/K_(M)) ofat least or about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000,6000, 7000, 8000, 9000, 10⁴, 10⁵, 10⁶ M⁻¹s⁻¹ or any range derivabletherein.

A modified polypeptide as discussed above may be characterized as havinga certain percentage of identity as compared to an unmodifiedpolypeptide (e.g., a native polypeptide) or to any polypeptide sequencedisclosed herein. For example, the unmodified polypeptide may compriseat least, or up to, about 150, 200, 250, 300, 350, 400 residues (or anyrange derivable therein) of a native kynureninase. The percentageidentity may be about, at most or at least 40%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any rangederivable therein) between the modified and unmodified polypeptides, orbetween any two sequences in comparison. It is also contemplated thatpercentage of identity discussed above may relate to a particularmodified region of a polypeptide as compared to an unmodified region ofa polypeptide. For instance, a polypeptide may contain a modified ormutant substrate recognition site of a kynureninase that can becharacterized based on the identity of the amino acid sequence of themodified or mutant substrate recognition site of the kynureninase tothat of an unmodified or mutant kynureninase from the same species oracross the species. A modified or mutant human polypeptidecharacterized, for example, as having at least 90% identity to anunmodified kynureninase means that at least 90% of the amino acids inthat modified or mutant human polypeptide are identical to the aminoacids in the unmodified polypeptide.

Such an unmodified polypeptide may be a native kynureninase,particularly a human isoform or other primate isoforms. For example, thenative human kynureninase may have the sequence of SEQ ID NO: 8.Non-limiting examples of other native primate kynureninase include Pongoabelii kynureninase (Genbank ID: XP_002812508.1; SEQ ID NO: 10), Macacafascicularis kynureninase (Genbank ID: EHH54849.1; SEQ ID NO: 11), andPan troglodytes kynureninase (Genbank ID: XP_003309314.1; SEQ ID NO:12). Exemplary native polypeptides include a sequence having about, atmost or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identity (or any range derivable therein) of SEQID NO: 8 or 10-12 or a fragment thereof. For example, the nativepolypeptide may comprise at least or up to about 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 415 residues (or anyrange derivable therein) of the sequence of SEQ ID NO: 8 or 10-12.

In some embodiments, the native kynureninase may be modified by one ormore other modifications, such as chemical modifications, substitutions,insertions, deletions, and/or truncations. For example, themodifications may be at a substrate recognitions site of the nativeenzyme. In a particular embodiment, the native kynureninase may bemodified by substitutions. For example, the number of substitutions maybe one, two, three, four or more. In further embodiments, the nativekynureninase may be modified in the substrate recognition site or anylocation that may affect substrate specificity.

In one embodiment, an isolated, modified human kynureninase enzyme isprovided, wherein the modified enzyme has at least one substitutionrelative to native human kynureninase (see SEQ ID NO: 8), and whereinthe at least one substitution includes a Met or Leu substitution for aPhe normally found at position 306 of native human kynureninase. Thus,in one aspect, an isolated, modified human kynureninase enzyme isprovided that comprises a Phe306Met substitution. In another aspect, anisolated, modified human kynureninase enzyme is provided that comprisesa Phe306Leu substitution.

In some aspects, the present invention also contemplates polypeptidescomprising a kynureninase linked to a heterologous amino acid sequence.For example, the kynureninase may be linked to the heterologous aminoacid sequence as a fusion protein. In a particular embodiment, thekynureninase may be linked to amino acid sequences, such as an IgG Fc,albumin, an albumin binding protein, or an XTEN polypeptide forincreasing the in vivo half-life.

To increase serum stability, the kynureninase may be linked to one ormore polyether molecules. In a particular embodiment, the polyether maybe polyethylene glycol (PEG). The polypeptide may be linked (e.g.,covalently) to PEG via specific amino acid residues, such as lysine orcysteine. For therapeutic administration, such a polypeptide comprisingthe kynureninase may be dispersed in a pharmaceutically acceptablecarrier.

In some aspects, a nucleic acid encoding such a kynureninase iscontemplated. In some embodiments, the nucleic acid has been codonoptimized for expression in bacteria. In particular embodiments, thebacteria is E. coli. In other aspects, the nucleic acid has been codonoptimized for expression in fungus (e.g., yeast), insects, or mammals.The present invention further contemplates vectors, such as expressionvectors, containing such nucleic acids. In particular embodiments, thenucleic acid encoding the kynureninase is operably linked to a promoter,including but not limited to heterologous promoters. In one embodiment,a kynureninase may be delivered to a target cell by a vector (e.g., agene therapy vector). Such viruses may have been modified by recombinantDNA technology to enable the expression of the kynureninase-encodingnucleic acid in the target cell. These vectors may be derived fromvectors of non-viral (e.g., plasmids) or viral (e.g., adenovirus,adeno-associated virus, retrovirus, lentivirus, herpes virus, orvaccinia virus) origin. Non-viral vectors are preferably complexed withagents to facilitate the entry of the DNA across the cellular membrane.Examples of such non-viral vector complexes include the formulation withpolycationic agents which facilitate the condensation of the DNA andlipid-based delivery systems. An example of a lipid-based deliverysystem would include liposome based delivery of nucleic acids.

In still further aspects, the present invention further contemplateshost cells comprising such vectors. The host cells may be bacteria(e.g., E. coli), fungal cells (e.g., yeast), insect cells, or mammaliancells.

In some embodiments, the vectors are introduced into host cells forexpressing the kynureninase. The proteins may be expressed in anysuitable manner. In one embodiment, the proteins are expressed in a hostcell such that the protein is glycosylated. In another embodiment, theproteins are expressed in a host cell such that the protein isaglycosylated.

Certain aspects of the present invention also contemplate methods oftreatment by the administration of the kynureninase peptide, the nucleicacid encoding the kynureninase in a gene therapy vector, or theformulation of the present invention, and in particular methods oftreating tumor cells or subjects with cancer. The subject may be anyanimal, such as a mouse. For example, the subject may be a mammal,particularly a primate, and more particularly a human patient. In someembodiments, the method may comprise selecting a patient with cancer.

In some embodiments, the cancer is any cancer that is sensitive tokynurenine depletion. In one embodiment, the present inventioncontemplates a method of treating a tumor cell or a cancer patientcomprising administering a formulation comprising such a polypeptide. Insome embodiments, the administration occurs under conditions such thatat least a portion of the cells of the cancer are killed. In anotherembodiment, the formulation comprises such a kynureninase withkynurenine-degrading activity at physiological conditions and furthercomprising an attached polyethylene glycol chain. In some embodiment,the formulation is a pharmaceutical formulation comprising any of theabove discussed kynureninases and pharmaceutically acceptableexcipients. Such pharmaceutically acceptable excipients are well knownto those of skill in the art. All of the above kynureninases may becontemplated as useful for human therapy.

In a further embodiment, there may also be provided a method of treatinga tumor cell comprising administering a formulation comprising anon-bacterial (mammalian, e.g., primate or mouse) kynureninase that haskynurenine-degrading activity or a nucleic acid encoding thereof.

The administration or treatment may be directed to the nutrient sourcefor the cells, and not necessarily the cells themselves. Therefore, inan in vivo application, treating a tumor cell includes contacting thenutrient medium for a population of tumor cells with the kynureninase.In this embodiment, the medium can be blood, lymphatic fluid, spinalfluid and the like bodily fluid where kynurenine depletion is desired.

In accordance with certain aspects of the present invention, such aformulation containing the kynureninase can be administeredintravenously, intradermally, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostaticaly,intrapleurally, intrasynovially, intratracheally, intranasally,intravitreally, intravaginally, intrarectally, intratumorally,intramuscularly, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularly, orally,topically, by inhalation, infusion, continuous infusion, localizedperfusion, via a catheter, via a lavage, in lipid compositions (e.g.,liposomes), or by other method or any combination of the forgoing aswould be known to one of ordinary skill in the art.

In a further embodiment, the method may also comprise administering atleast a second anticancer therapy to the subject. The second anticancertherapy may be a surgical therapy, chemotherapy, radiation therapy,cryotherapy, hormone therapy, immunotherapy or cytokine therapy. Incertain aspects, the second anticancer therapy may be an anti-PD-1,anti-CTLA-4, or anti-PD-L1 antibody.

In some embodiment, a cell comprising a chimeric antigen receptor (CAR)and a kynureninase enzyme are contemplated for use in treating a subjectwith cancer. In some aspects, the cell may be transfected with a DNAencoding the CAR and the kynureninase and, in some cases, a transposase.

The CAR may target any cancer-cell antigen of interest, including, forexample, HER2, CD19, CD20, and GD2. The antigen binding regions ordomain can comprise a fragment of the V_(H) and V_(L) chains of asingle-chain variable fragment (scFv) derived from a particular humanmonoclonal antibody, such as those described in U.S. Pat. No. 7,109,304,which is incorporated herein by reference in its entirety. The fragmentcan also be any number of different antigen binding domains of a humanantigen-specific antibody. In a more specific embodiment, the fragmentis an antigen-specific scFv encoded by a sequence that is optimized forhuman codon usage for expression in human cells. For additional examplesof CARs, see, for example, WO 2012/031744, WO 2012/079000, WO2013/059593, and U.S. Pat. No. 8,465,743, all of which are incorporatedherein by reference in their entireties.

The kynureninase may be any kynureninase disclosed herein. Methods oftransfecting of cells are well known in the art, but in certain aspects,highly efficient transfections methods such as electroporation areemployed. For example, nucleic acids may be introduced into cells usinga nucleofection apparatus. Preferably, the transfection step does notinvolve infecting or transducing the cells with virus, which can causegenotoxicity and/or lead to an immune response to cells containing viralsequences in a treated subject.

A wide range of CAR constructs and expression vectors for the same areknown in the art and are further detailed herein. For example, in someaspects, the CAR expression vector is a DNA expression vector such as aplasmid, linear expression vector or an episome. In some aspects, thevector comprises additional sequences, such as sequence that facilitatesexpression of the CAR, such a promoter, enhancer, poly-A signal, and/orone or more introns. In preferred aspects, the CAR coding sequence isflanked by transposon sequences, such that the presence of a transposaseallows the coding sequence to integrate into the genome of thetransfected cell.

In certain aspects, cells are further transfected with a transposasethat facilitates integration of a CAR coding sequence into the genome ofthe transfected cells. In some aspects, the transposase is provided asDNA expression vector. However, in preferred aspects, the transposase isprovided as an expressible RNA or a protein such that long-termexpression of the transposase does not occur in the transgenic cells.Any transposase system may be used in accordance with the embodiments.In other aspects, cells may be infected with a lentivirus to facilitateintegration of the CAR coding sequence and the kynureninase codingsequence into the genome of the cell.

In one embodiment, a composition comprising a kynureninase or a nucleicacid encoding a kynureninase is provided for use in the treatment of atumor in a subject. In another embodiment, the use of a kynureninase ora nucleic acid encoding a kynureninase in the manufacture of amedicament for the treatment of a tumor is provided. Said kynureninasemay be any kynureninase of the embodiments.

Embodiments discussed in the context of methods and/or compositions ofthe invention may be employed with respect to any other method orcomposition described herein. Thus, an embodiment pertaining to onemethod or composition may be applied to other methods and compositionsof the invention as well.

As used herein the terms “encode” or “encoding,” with reference to anucleic acid, are used to make the invention readily understandable bythe skilled artisan; however, these terms may be used interchangeablywith “comprise” or “comprising,” respectively.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1—SDS-PAGE of (lane 1) PRECISION PLUS PROTEIN™ MW standard (BioRad)(lanes 2-4) increasing concentrations of Pf-KYNU and (lane 5) PEG 5,000MW modified Pf-KYNU.

FIG. 2—Stability of Pf-KYNU in (open square) PBS and (open circle)pooled human serum.

FIG. 3—Efficacy of PEG-Pf-KYNU in an autologous B16 mouse melanoma modelas measured by tumor growth rates. (Solid square) Heat inactivatedPEG-Pf-KYNU. (Solid circle) Active PEG-Pf-KYNU.

FIG. 4—Efficacy of PEG-Pf-KYNU in an autologous B16 mouse melanoma modelas measured by survival. (Solid square) Heat inactivated PEG-Pf-KYNU.(Solid circle) Active PEG-Pf-KYNU.

FIGS. 5A-5B—Mice treated with heat-inactivated PEG-Pf-KYNU. (•) Micetreated with active PEG-Pf-KYNU. FIG. 5A—The population of circulatingCD4+ regulatory T-cell is significantly smaller in the group treatedwith active PEG-Pf-KYNU. FIG. 5B—The population of tumor infiltratingCD8+ T-cells shows significantly higher expression of granzyme B andinterferon γ.

FIG. 6—Genetic selection for kynureninase activity in E. coli. E.coli-ΔtrpE cells plated on M9 minimal media plates with filter paperdisks soaked in L-Trp (Trp), buffer (-), anthranilic acid (AA), or L-Kyn(Kyn).

FIG. 7—In vitro stability of Mucilaginibacter paludis kynureninase(Mu-KYNU). Activity as a function of time of Mu-KYNU (open square) inPBS at 37° C. with a ¹T_(1/2)=6 h with an amplitude of 74% remainingactivity and a subsequent ²T_(1/2)=150 h, and (solid circle) in pooledhuman serum at 37° C. with a ¹T_(1/2)=5 h with an amplitude of 30%remaining activity and a subsequent ²T_(1/2)=73 h.

FIG. 8—Kaplan-Meier plot of B16 allografts in C57BL/6J treated witheither PEG-Pf-KYNU (--●), deactivated PEG-Pf-KYNU (-●●), anti-PD1 (●●●),or anti-CTLA-4 (▬). Arrows indicate treatment days, (A) indicatestreatment with antibody, (E) indicates treatment with enzyme.

FIGS. 9A-9C—FIG. 9A—C57BL/6J bearing B16 tumor allografts treated withPBS (circle) (control), anti-PD1 alone (square), anti-PD1/PEG-Mu-KYNU(upside-down triangle), or anti-PD1/PEG-Pf-KYNU (right-side uptriangle). FIG. 9B—Additive effects were observed withanti-PD1/PEG-Pf-KYNU combination treatment eliminating 60% of tumors andanti-PD1/PEG-Mu-KYNU combination eliminating 20% of tumors compared to0% tumor elimination with anti-PD1 alone. FIG. 9C—CorrespondingKaplan-Meier plot.

FIGS. 10A-10B—FIG. 10A—C57BL/6J bearing B16 tumor allografts treatedwith heat-inactivated PEG-Mu-KYNU (▪) or active PEG-Mu-KYNU (▴). FIG.10B—Corresponding Kaplan-Meier plot depicting a median survival time of25 days for PEG-Mu-KYNU (---), and median survival time of 22 days forheat-inactivated PEG-Mu-KYNU (▬) (arrows indicate treatment days).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Kynurenine is a metabolite of the amino acid tryptophan generated viathe action of either indolamine-2,3-dioxygenase (IDO) ortryptophan-2,3-dioxygenase (TDO). Kynurenine exerts multiple effects oncell physiology, one of the most important of which is modulation of Tcell responses. Many tumor cells regulate the synthesis of IDO and/orTDO to elevate the local concentration of kynurenine, which isaccompanied with depletion of tryptophan. High levels of kynurenineserve as a powerful way to inhibit the function of tumor infiltrating Tcells that would otherwise attack the tumor.

The present invention provides methods for the use of kynureninedegrading enzymes as a means for depleting local kynurenine levels inthe tumor microenvironment as well as in the serum and thus preventtumor-mediated suppression of T-cell action. Kynurenine hydrolyzingenzymes (kynureninases) convert kynurenine to alanine and anthranilicacid, the latter of which is not known to affect T-cell function. Theinventors generated a pharmaceutical preparation of kynureninase enzymeto enable the enzyme to persist for prolonged times under physiologicalconditions. The inventors then showed that intratumoral administrationof the enzyme results in dramatic retardation of growth of an aggressivetumor in mice.

I. DEFINITIONS

As used herein the terms “protein” and “polypeptide” refer to compoundscomprising amino acids joined via peptide bonds and are usedinterchangeably.

As used herein, the term “fusion protein” refers to a chimeric proteincontaining proteins or protein fragments operably linked in a non-nativeway.

As used herein, the term “half-life” (½-life) refers to the time thatwould be required for the concentration of a polypeptide thereof to fallby half in vitro or in vivo, for example, after injection in a mammal.

The terms “in operable combination,” “in operable order,” and “operablylinked” refer to a linkage wherein the components so described are in arelationship permitting them to function in their intended manner, forexample, a linkage of nucleic acid sequences in such a manner that anucleic acid molecule capable of directing the transcription of a givengene and/or the synthesis of desired protein molecule, or a linkage ofamino acid sequences in such a manner so that a fusion protein isproduced.

The term “linker” is meant to refer to a compound or moiety that acts asa molecular bridge to operably link two different molecules, wherein oneportion of the linker is operably linked to a first molecule, andwherein another portion of the linker is operably linked to a secondmolecule.

The term “PEGylated” refers to conjugation with polyethylene glycol(PEG), which has been widely used as a drug carrier, given its highdegree of biocompatibility and ease of modification. PEG can be coupled(e.g., covalently linked) to active agents through the hydroxy groups atthe end of the PEG chain via chemical methods; however, PEG itself islimited to at most two active agents per molecule. In a differentapproach, copolymers of PEG and amino acids have been explored as novelbiomaterial that would retain the biocompatibility of PEG, but thatwould have the added advantage of numerous attachment points permolecule (thus providing greater drug loading), and that can besynthetically designed to suit a variety of applications.

The term “gene” refers to a DNA sequence that comprises control andcoding sequences necessary for the production of a polypeptide orprecursor thereof. The polypeptide can be encoded by a full-lengthcoding sequence or by any portion of the coding sequence so as thedesired enzymatic activity is retained.

The term “native” refers to the typical form of a gene, a gene product,or a characteristic of that gene or gene product when isolated from anaturally occurring source. A native form is that which is mostfrequently observed in a natural population and is thus arbitrarilydesignated the normal or wild-type form. In contrast, the term“modified,” “variant,” or “mutant” refers to a gene or gene product thatdisplays modification in sequence and functional properties (i.e.,altered characteristics) when compared to the native gene or geneproduct.

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques (see, for example, Maniatis et al., 1988 and Ausubel et al.,1994, both incorporated herein by reference).

The term “expression vector” refers to any type of genetic constructcomprising a nucleic acid coding for an RNA capable of beingtranscribed. In some cases, RNA molecules are then translated into aprotein, polypeptide, or peptide. In other cases, these sequences arenot translated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host cell. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

The term “therapeutically effective amount” as used herein refers to anamount of cells and/or therapeutic composition (such as a therapeuticpolynucleotide and/or therapeutic polypeptide) that is employed inmethods to achieve a therapeutic effect. The term “therapeutic benefit”or “therapeutically effective” as used throughout this applicationrefers to anything that promotes or enhances the well-being of thesubject with respect to the medical treatment of this condition. Thisincludes, but is not limited to, a reduction in the frequency orseverity of the signs or symptoms of a disease. For example, treatmentof cancer may involve, for example, a reduction in the size of a tumor,a reduction in the invasiveness of a tumor, reduction in the growth rateof the cancer, or prevention of metastasis. Treatment of cancer may alsorefer to prolonging survival of a subject with cancer.

The term “K_(M)” as used herein refers to the Michaelis-Menten constantfor an enzyme and is defined as the concentration of the specificsubstrate at which a given enzyme yields one-half its maximum velocityin an enzyme catalyzed reaction. The term “k_(cat)” as used hereinrefers to the turnover number or the number of substrate molecules eachenzyme site converts to product per unit time, and in which the enzymeis working at maximum efficiency. The term “k_(cat)/K_(M)” as usedherein is the specificity constant, which is a measure of howefficiently an enzyme converts a substrate into product.

The term “chimeric antigen receptors (CARs),” as used herein, may referto artificial T-cell receptors, chimeric T-cell receptors, or chimericimmunoreceptors, for example, and encompass engineered receptors thatgraft an artificial specificity onto a particular immune effector cell.CARs may be employed to impart the specificity of a monoclonal antibodyonto a T cell, thereby allowing a large number of specific T cells to begenerated, for example, for use in adoptive cell therapy. In specificembodiments, CARs direct specificity of the cell to a tumor associatedantigen, for example. In some embodiments, CARs comprise anintracellular activation domain, a transmembrane domain, and anextracellular domain comprising a tumor associated antigen bindingregion. In particular aspects, CARs comprise fusions of single-chainvariable fragments (scFv) derived from monoclonal antibodies (such asthose described in U.S. Pat. No. 7,109,304, which is incorporated hereinby reference in its entirety), fused to CD3-zeta transmembrane andendodomains. The specificity of other CAR designs may be derived fromligands of receptors (e.g., peptides) or from pattern-recognitionreceptors, such as Dectins. In particular embodiments, one can targetmalignant B cells by redirecting the specificity of T cells by using aCAR specific for the B-lineage molecule, CD19. In certain cases, thespacing of the antigen-recognition domain can be modified to reduceactivation-induced cell death. In certain cases, CARs comprise domainsfor additional co-stimulatory signaling, such as CD3-zeta, FcR, CD27,CD28, CD137, DAP10, and/or OX40. In some cases, molecules can beco-expressed with the CAR, including co-stimulatory molecules, reportergenes for imaging (e.g., for positron emission tomography), geneproducts that conditionally ablate the T cells upon addition of apro-drug, homing receptors, chemokines, chemokine receptors, cytokines,and cytokine receptors.

“Treatment” and “treating” refer to administration or application of atherapeutic agent to a subject or performance of a procedure or modalityon a subject for the purpose of obtaining a therapeutic benefit of adisease or health-related condition. For example, a treatment mayinclude administration of a pharmaceutically effective amount of akynureninase.

“Subject” and “patient” refer to either a human or non-human, such asprimates, mammals, and vertebrates. In particular embodiments, thesubject is a human.

II. KYNURENINASE POLYPEPTIDES

Some embodiments concern modified proteins and polypeptides. Particularembodiments concern a modified protein or polypeptide that exhibits atleast one functional activity that is comparable to the unmodifiedversion, preferably, the kynurenine degrading activity or the3′-hydroxy-kynurenine degrading activity. In further aspects, theprotein or polypeptide may be further modified to increase serumstability. Thus, when the present application refers to the function oractivity of “modified protein” or a “modified polypeptide,” one ofordinary skill in the art would understand that this includes, forexample, a protein or polypeptide that possesses an additional advantageover the unmodified protein or polypeptide, such as kynurenine degradingactivity or 3′-hydroxy-kynurenine degrading activity. In certainembodiments, the unmodified protein or polypeptide is a nativekynureninase, preferably a human kynureninase or the Pseudomonasfluorescens kynureninase. It is specifically contemplated thatembodiments concerning a “modified protein” may be implemented withrespect to a “modified polypeptide,” and vice versa.

Determination of activity may be achieved using assays familiar to thoseof skill in the art, particularly with respect to the protein'sactivity, and may include for comparison purposes, the use of nativeand/or recombinant versions of either the modified or unmodified proteinor polypeptide.

In certain embodiments, a modified polypeptide, such as a modifiedkynureninase, may be identified based on its increase in kynurenineand/or 3′-hydroxy-kynurenine degrading activity. For example, substraterecognition sites of the unmodified polypeptide may be identified. Thisidentification may be based on structural analysis or homology analysis.A population of mutants involving modifications of such substraterecognition sites may be generated. In a further embodiment, mutantswith increased kynurenine degrading activity may be selected from themutant population. Selection of desired mutants may include methods,such as detection of byproducts or products from kynurenine degradation.

Modified proteins may possess deletions and/or substitutions of aminoacids; thus, a protein with a deletion, a protein with a substitution,and a protein with a deletion and a substitution are modified proteins.In some embodiments, these modified proteins may further includeinsertions or added amino acids, such as with fusion proteins orproteins with linkers, for example. A “modified deleted protein” lacksone or more residues of the native protein, but may possess thespecificity and/or activity of the native protein. A “modified deletedprotein” may also have reduced immunogenicity or antigenicity. Anexample of a modified deleted protein is one that has an amino acidresidue deleted from at least one antigenic region that is, a region ofthe protein determined to be antigenic in a particular organism, such asthe type of organism that may be administered the modified protein.

Substitution or replacement variants typically contain the exchange ofone amino acid for another at one or more sites within the protein andmay be designed to modulate one or more properties of the polypeptide,particularly its effector functions and/or bioavailability.Substitutions may or may not be conservative, that is, one amino acid isreplaced with one of similar shape and charge. Conservativesubstitutions are well known in the art and include, for example, thechanges of: alanine to serine; arginine to lysine; asparagine toglutamine or histidine; aspartate to glutamate; cysteine to serine;glutamine to asparagine; glutamate to aspartate; glycine to proline;histidine to asparagine or glutamine; isoleucine to leucine or valine;leucine to valine or isoleucine; lysine to arginine; methionine toleucine or isoleucine; phenylalanine to tyrosine, leucine, ormethionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine.

In addition to a deletion or substitution, a modified protein maypossess an insertion of residues, which typically involves the additionof at least one residue in the polypeptide. This may include theinsertion of a targeting peptide or polypeptide or simply a singleresidue. Terminal additions, called fusion proteins, are discussedbelow.

The term “biologically functional equivalent” is well understood in theart and is further defined in detail herein. Accordingly, sequences thathave between about 70% and about 80%, or between about 81% and about90%, or even between about 91% and about 99% of amino acids that areidentical or functionally equivalent to the amino acids of a controlpolypeptide are included, provided the biological activity of theprotein is maintained. A modified protein may be biologicallyfunctionally equivalent to its native counterpart in certain aspects.

It also will be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to nucleic acidsequences that may, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

III. ENZYMATIC KYNURENINE DEGRADATION FOR THERAPY

In certain aspects, the polypeptides may be used for the treatment ofdiseases, including cancers that are sensitive to kynurenine depletion,with enzymes that deplete kynurenine, to prevent tumor-mediatedtolerogenic effects and instead mediate tumor-ablating pro-inflammatoryresponses. In certain aspects, kynureninases are contemplated for use intreating tumors expressing IDO1, IDO2, and/or TDO.

Certain aspects of the present invention provide a modified kynureninasefor treating diseases, such as tumors. Particularly, the modifiedpolypeptide may have human polypeptide sequences and thus may preventallergic reactions in human patients, allow repeated dosing, andincrease the therapeutic efficacy.

Tumors for which the present treatment methods are useful include anymalignant cell type, such as those found in a solid tumor or ahematological tumor. Exemplary solid tumors can include, but are notlimited to, a tumor of an organ selected from the group consisting ofpancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney,larynx, sarcoma, lung, bladder, melanoma, prostate, and breast.Exemplary hematological tumors include tumors of the bone marrow, T or Bcell malignancies, leukemias, lymphomas, blastomas, myelomas, and thelike. Further examples of cancers that may be treated using the methodsprovided herein include, but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, leukemia, squamous cell cancer, lung cancer(including small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung), cancerof the peritoneum, hepatocellular cancer, gastric or stomach cancer(including gastrointestinal cancer and gastrointestinal stromal cancer),pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, breast cancer, colon cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, prostate cancer, vulval cancer, thyroid cancer, varioustypes of head and neck cancer, melanoma, superficial spreading melanoma,lentigo malignant melanoma, acral lentiginous melanomas, nodularmelanomas, as well as B-cell lymphoma (including low grade/follicularnon-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's macroglobulinemia), chroniclymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), Hairycell leukemia, multiple myeloma, acute myeloid leukemia (AML) andchronic myeloblastic leukemia.

The cancer may specifically be of the following histological type,though it is not limited to these: neoplasm, malignant; carcinoma;carcinoma, undifferentiated; giant and spindle cell carcinoma; smallcell carcinoma; papillary carcinoma; squamous cell carcinoma;lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma;transitional cell carcinoma; papillary transitional cell carcinoma;adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma andcholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma;adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposiscoli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolaradenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clearcell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;papillary and follicular adenocarcinoma; nonencapsulating sclerosingcarcinoma; adrenal cortical carcinoma; endometroid carcinoma; skinappendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma;ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma;papillary cystadenocarcinoma; papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cellcarcinoma; infiltrating duct carcinoma; medullary carcinoma; lobularcarcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cellcarcinoma; adenosquamous carcinoma; adenocarcinoma w/squamousmetaplasia; thymoma, malignant; ovarian stromal tumor, malignant;thecoma, malignant; granulosa cell tumor, malignant; androblastoma,malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipidcell tumor, malignant; paraganglioma, malignant; extra-mammaryparaganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignantmelanoma; amelanotic melanoma; superficial spreading melanoma; malignantmelanoma in giant pigmented nevus; epitheloid cell melanoma; blue nevus,malignant; sarcoma; fibrosarcoma; fibrous hi stiocytoma, malignant;myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonalrhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixedtumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma;carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant;phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant;dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii,malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma;hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma,malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma;chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma;giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant;ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblasticfibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant;ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillaryastrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignantlymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse;malignant lymphoma, follicular; mycosis fungoides; other specifiednon-hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mastcell sarcoma; immunoproliferative small intestinal disease; leukemia;lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcomacell leukemia; myeloid leukemia; basophilic leukemia; eosinophilicleukemia; monocytic leukemia; mast cell leukemia; megakaryoblasticleukemia; myeloid sarcoma; and hairy cell leukemia.

The kynureninase may be used herein as an antitumor agent in a varietyof modalities for depleting kynurenine and/or 3′-hydroxy-kynurenine fromtumor tissue, or the circulation of a mammal with cancer, or fordepletion of kynurenine where its depletion is considered desirable.

Depletion can be conducted in vivo in the circulation of a mammal, invitro in cases where kynurenine and 3′-hydroxy-kynurenine depletion intissue culture or other biological mediums is desired, and in ex vivoprocedures where biological fluids, cells, or tissues are manipulatedoutside the body and subsequently returned to the body of the patientmammal. Depletion of kynurenine from circulation, culture media,biological fluids, or cells is conducted to reduce the amount ofkynurenine accessible to the material being treated, and thereforecomprises contacting the material to be depleted with akynurenine-depleting amount of the kynureninase underkynurenine-depleting conditions as to degrade the ambient kynurenine inthe material being contacted.

The depletion may be directed to the nutrient source for the cells, andnot necessarily the cells themselves. Therefore, in an in vivoapplication, treating a tumor cell includes contacting the nutrientmedium for a population of tumor cells with the kynureninase. In thisembodiment, the medium may be blood, lymphatic fluid, spinal fluid andthe like bodily fluid where kynurenine depletion is desired.

Kynurenine- and 3′-hydroxy-kynurenine-depleting efficiency can varywidely depending upon the application, and typically depends upon theamount of kynurenine present in the material, the desired rate ofdepletion, and the tolerance of the material for exposure tokynureninase. Kynurenine and kynurenine metabolite levels in a material,and therefore rates of kynurenine and kynurenine metabolite depletionfrom the material, can readily be monitored by a variety of chemical andbiochemical methods well known in the art. Exemplarykynurenine-depleting amounts are described further herein, and can rangefrom 0.001 to 100 units (U) of kynureninase, preferably about 0.01 to 10U, and more preferably about 0.1 to 5 U kyureninase per milliliter (mL)of material to be treated. Typical dosages can be administered based onbody weight, and are in the range of about 5-1000 U/kilogram (kg)/day,preferably about 5-100 U/kg/day, more preferably about 10-50 U/kg/day,and more preferably about 20-40 U/kg/day.

Kynurenine-depleting conditions are buffer and temperature conditionscompatible with the biological activity of a kynureninase, and includemoderate temperature, salt, and pH conditions compatible with theenzyme, for example, physiological conditions. Exemplary conditionsinclude about 4-40° C., ionic strength equivalent to about 0.05 to 0.2 MNaCl, and a pH of about 5 to 9, while physiological conditions areincluded.

In a particular embodiment, the invention contemplates methods of usinga kynureninase as an antitumor agent, and therefore comprises contactinga population of tumor cells with a therapeutically effective amount ofkynureninase for a time period sufficient to inhibit tumor cell growth.

In one embodiment, the contacting in vivo is accomplished byadministering, by intravenous intraperitoneal, or intratumoralinjection, a therapeutically effective amount of a physiologicallytolerable composition comprising an kynureninase of this invention to apatient, thereby depleting the kynurenine source of the tumor cellspresent in the patient.

A therapeutically effective amount of a kynureninase is a predeterminedamount calculated to achieve the desired effect, i.e., to depletekynurenine in the tumor tissue or in a patient's circulation, andthereby mediate a tumor-ablating pro-inflammatory response. Thus, thedosage ranges for the administration of kynureninase of the inventionare those large enough to produce the desired effect in which thesymptoms of tumor cell division and cell cycling are reduced. The dosageshould not be so large as to cause adverse side effects, such ashyperviscosity syndromes, pulmonary edema, congestive heart failure,neurological effects, and the like. Generally, the dosage will vary withage of, condition of, sex of, and extent of the disease in the patientand can be determined by one of skill in the art. The dosage can beadjusted by the individual physician in the event of any complication.

The kynureninase can be administered parenterally by injection or bygradual infusion over time. The kynureninase can be administeredintravenously, intraperitoneally, orally, intramuscularly,subcutaneously, intracavity, transdermally, dermally, can be deliveredby peristaltic means, can be injected directly into the tissuecontaining the tumor cells, or can be administered by a pump connectedto a catheter that may contain a potential biosensor for kynurenine.

The therapeutic compositions containing kynureninase are conventionallyadministered intravenously, as by injection of a unit dose, for example.The term “unit dose” when used in reference to a therapeutic compositionrefers to physically discrete units suitable as unitary dosage for thesubject, each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect inassociation with the required diluent, i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's system to utilize the active ingredient, and degree oftherapeutic effect desired. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitionerand are peculiar to each individual. However, suitable dosage ranges forsystemic application are disclosed herein and depend on the route ofadministration. Suitable regimes for initial administration and boostershots are also contemplated and are typified by an initialadministration followed by repeated doses at one or more hour intervalsby a subsequent injection or other administration. Exemplary multipleadministrations are described herein and are particularly preferred tomaintain continuously high serum and tissue levels of kynureninase andconversely low serum and tissue levels of kynurenine. Alternatively,continuous intravenous infusion sufficient to maintain concentrations inthe blood in the ranges specified for in vivo therapies arecontemplated.

IV. CONJUGATES

Compositions and methods of the present invention involve modifiedkynureninases, such as by forming conjugates with heterologous peptidesegments or polymers, such as polyethylene glycol. In further aspects,the kynureninases may be linked to PEG to increase the hydrodynamicradius of the enzyme and hence increase the serum persistence. Incertain aspects, the disclosed polypeptide may be conjugated to anytargeting agent, such as a ligand having the ability to specifically andstably bind to an external receptor or binding site on a tumor cell(U.S. Patent Publ. 2009/0304666).

A. Fusion Proteins

Certain embodiments of the present invention concern fusion proteins.These molecules may have a native or modified kynureninase linked at theN- or C-terminus to a heterologous domain. For example, fusions may alsoemploy leader sequences from other species to permit the recombinantexpression of a protein in a heterologous host. Another useful fusionincludes the addition of a protein affinity tag, such as a serum albuminaffinity tag or six histidine residues, or an immunologically activedomain, such as an antibody epitope, preferably cleavable, to facilitatepurification of the fusion protein. Non-limiting affinity tags includepolyhistidine, chitin binding protein (CBP), maltose binding protein(MBP), and glutathione-S-transferase (GST).

In a particular embodiment, the kynureninase may be linked to a peptidethat increases the in vivo half-life, such as an XTEN polypeptide(Schellenberger et al., 2009), IgG Fc domain, albumin, or albuminbinding peptide.

Methods of generating fusion proteins are well known to those of skillin the art. Such proteins can be produced, for example, by de novosynthesis of the complete fusion protein, or by attachment of the DNAsequence encoding the heterologous domain, followed by expression of theintact fusion protein.

Production of fusion proteins that recover the functional activities ofthe parent proteins may be facilitated by connecting genes with abridging DNA segment encoding a peptide linker that is spliced betweenthe polypeptides connected in tandem. The linker would be of sufficientlength to allow proper folding of the resulting fusion protein.

B. Linkers

In certain embodiments, the kynureninase may be chemically conjugatedusing bifunctional cross-linking reagents or fused at the protein levelwith peptide linkers.

Bifunctional cross-linking reagents have been extensively used for avariety of purposes, including preparation of affinity matrices,modification and stabilization of diverse structures, identification ofligand and receptor binding sites, and structural studies. Suitablepeptide linkers may also be used to link the kynureninase, such asGly-Ser linkers.

Homobifunctional reagents that carry two identical functional groupsproved to be highly efficient in inducing cross-linking betweenidentical and different macromolecules or subunits of a macromolecule,and linking of polypeptide ligands to their specific binding sites.Heterobifunctional reagents contain two different functional groups. Bytaking advantage of the differential reactivities of the two differentfunctional groups, cross-linking can be controlled both selectively andsequentially. The bifunctional cross-linking reagents can be dividedaccording to the specificity of their functional groups, e.g., amino-,sulfhydryl-, guanidine-, indole-, carboxyl-specific groups. Of these,reagents directed to free amino groups have become especially popularbecause of their commercial availability, ease of synthesis, and themild reaction conditions under which they can be applied.

A majority of heterobifunctional cross-linking reagents contain aprimary amine-reactive group and a thiol-reactive group. In anotherexample, heterobifunctional cross-linking reagents and methods of usingthe cross-linking reagents are described (U.S. Pat. No. 5,889,155,specifically incorporated herein by reference in its entirety). Thecross-linking reagents combine a nucleophilic hydrazide residue with anelectrophilic maleimide residue, allowing coupling, in one example, ofaldehydes to free thiols. The cross-linking reagent can be modified tocross-link various functional groups.

Additionally, any other linking/coupling agents and/or mechanisms knownto those of skill in the art may be used to combine kynureninase, suchas, for example, antibody-antigen interaction, avidin biotin linkages,amide linkages, ester linkages, thioester linkages, ether linkages,thioether linkages, phosphoester linkages, phosphoramide linkages,anhydride linkages, disulfide linkages, ionic and hydrophobicinteractions, bispecific antibodies and antibody fragments, orcombinations thereof.

It is preferred that a cross-linker having reasonable stability in bloodwill be employed. Numerous types of disulfide-bond containing linkersare known that can be successfully employed to conjugate targeting andtherapeutic/preventative agents. Linkers that contain a disulfide bondthat is sterically hindered may prove to give greater stability in vivo.These linkers are thus one group of linking agents.

In addition to hindered cross-linkers, non-hindered linkers also can beemployed in accordance herewith. Other useful cross-linkers, notconsidered to contain or generate a protected disulfide, include SATA,SPDP, and 2-iminothiolane (Wawrzynczak and Thorpe, 1987). The use ofsuch cross-linkers is well understood in the art. Another embodimentinvolves the use of flexible linkers.

Once chemically conjugated, the peptide generally will be purified toseparate the conjugate from unconjugated agents and from othercontaminants. A large number of purification techniques are availablefor use in providing conjugates of a sufficient degree of purity torender them clinically useful.

Purification methods based upon size separation, such as gel filtration,gel permeation, or high performance liquid chromatography, willgenerally be of most use. Other chromatographic techniques, such asBlue-Sepharose separation, may also be used. Conventional methods topurify the fusion proteins from inclusion bodies may be useful, such asusing weak detergents, such as sodium N-lauroyl-sarcosine (SLS).

C. PEGylation

In certain aspects of the invention, methods and compositions related toPEGylation of kynureninase are disclosed. For example, the kynureninasemay be PEGylated in accordance with the methods disclosed herein.

PEGylation is the process of covalent attachment of poly(ethyleneglycol) polymer chains to another molecule, normally a drug ortherapeutic protein. PEGylation is routinely achieved by incubation of areactive derivative of PEG with the target macromolecule. The covalentattachment of PEG to a drug or therapeutic protein can “mask” the agentfrom the host's immune system (reduced immunogenicity and antigenicity)or increase the hydrodynamic size (size in solution) of the agent, whichprolongs its circulatory time by reducing renal clearance. PEGylationcan also provide water solubility to hydrophobic drugs and proteins.

The first step of the PEGylation is the suitable functionalization ofthe PEG polymer at one or both terminals. PEGs that are activated ateach terminus with the same reactive moiety are known as“homobifunctional,” whereas if the functional groups present aredifferent, then the PEG derivative is referred as “heterobifunctional”or “heterofunctional.” The chemically active or activated derivatives ofthe PEG polymer are prepared to attach the PEG to the desired molecule.

The choice of the suitable functional group for the PEG derivative isbased on the type of available reactive group on the molecule that willbe coupled to the PEG. For proteins, typical reactive amino acidsinclude lysine, cysteine, histidine, arginine, aspartic acid, glutamicacid, serine, threonine, and tyrosine. The N-terminal amino group andthe C-terminal carboxylic acid can also be used.

The techniques used to form first generation PEG derivatives aregenerally reacting the PEG polymer with a group that is reactive withhydroxyl groups, typically anhydrides, acid chlorides, chloroformates,and carbonates. In the second generation PEGylation chemistry moreefficient functional groups, such as aldehyde, esters, amides, etc., aremade available for conjugation.

As applications of PEGylation have become more and more advanced andsophisticated, there has been an increase in need for heterobifunctionalPEGs for conjugation. These heterobifunctional PEGs are very useful inlinking two entities, where a hydrophilic, flexible, and biocompatiblespacer is needed. Preferred end groups for heterobifunctional PEGs aremaleimide, vinyl sulfones, pyridyl disulfide, amine, carboxylic acids,and NHS esters.

The most common modification agents, or linkers, are based on methoxyPEG (mPEG) molecules. Their activity depends on adding aprotein-modifying group to the alcohol end. In some instancespolyethylene glycol (PEG diol) is used as the precursor molecule. Thediol is subsequently modified at both ends in order to make a hetero- orhomo-dimeric PEG-linked molecule.

Proteins are generally PEGylated at nucleophilic sites, such asunprotonated thiols (cysteinyl residues) or amino groups. Examples ofcysteinyl-specific modification reagents include PEG maleimide, PEGiodoacetate, PEG thiols, and PEG vinylsulfone. All four are stronglycysteinyl-specific under mild conditions and neutral to slightlyalkaline pH but each has some drawbacks. The thioether formed with themaleimides can be somewhat unstable under alkaline conditions so theremay be some limitation to formulation options with this linker. Thecarbamothioate linkage formed with iodo PEGs is more stable, but freeiodine can modify tyrosine residues under some conditions. PEG thiolsform disulfide bonds with protein thiols, but this linkage can also beunstable under alkaline conditions. PEG-vinylsulfone reactivity isrelatively slow compared to maleimide and iodo PEG; however, thethioether linkage formed is quite stable. Its slower reaction rate alsocan make the PEG-vinylsulfone reaction easier to control.

Site-specific PEGylation at native cysteinyl residues is seldom carriedout, since these residues are usually in the form of disulfide bonds orare required for biological activity. On the other hand, site-directedmutagenesis can be used to incorporate cysteinyl PEGylation sites forthiol-specific linkers. The cysteine mutation must be designed such thatit is accessible to the PEGylation reagent and is still biologicallyactive after PEGylation.

Amine-specific modification agents include PEG NHS ester, PEG tresylate,PEG aldehyde, PEG isothiocyanate, and several others. All react undermild conditions and are very specific for amino groups. The PEG NHSester is probably one of the more reactive agents; however, its highreactivity can make the PEGylation reaction difficult to control on alarge scale. PEG aldehyde forms an imine with the amino group, which isthen reduced to a secondary amine with sodium cyanoborohydride. Unlikesodium borohydride, sodium cyanoborohydride will not reduce disulfidebonds. However, this chemical is highly toxic and must be handledcautiously, particularly at lower pH where it becomes volatile.

Due to the multiple lysine residues on most proteins, site-specificPEGylation can be a challenge. Fortunately, because these reagents reactwith unprotonated amino groups, it is possible to direct the PEGylationto lower-pK amino groups by performing the reaction at a lower pH.Generally the pK of the alpha-amino group is 1-2 pH units lower than theepsilon-amino group of lysine residues. By PEGylating the molecule at pH7 or below, high selectivity for the N-terminus frequently can beattained. However, this is only feasible if the N-terminal portion ofthe protein is not required for biological activity. Still, thepharmacokinetic benefits from PEGylation frequently outweigh asignificant loss of in vitro bioactivity, resulting in a product withmuch greater in vivo bioactivity regardless of PEGylation chemistry.

There are several parameters to consider when developing a PEGylationprocedure. Fortunately, there are usually no more than four or five keyparameters. The “design of experiments” approach to optimization ofPEGylation conditions can be very useful. For thiol-specific PEGylationreactions, parameters to consider include: protein concentration,PEG-to-protein ratio (on a molar basis), temperature, pH, reaction time,and in some instances, the exclusion of oxygen. (Oxygen can contributeto intermolecular disulfide formation by the protein, which will reducethe yield of the PEGylated product.) The same factors should beconsidered (with the exception of oxygen) for amine-specificmodification except that pH may be even more critical, particularly whentargeting the N-terminal amino group.

For both amine- and thiol-specific modifications, the reactionconditions may affect the stability of the protein. This may limit thetemperature, protein concentration, and pH. In addition, the reactivityof the PEG linker should be known before starting the PEGylationreaction. For example, if the PEGylation agent is only 70 percentactive, the amount of PEG used should ensure that only active PEGmolecules are counted in the protein-to-PEG reaction stoichiometry.

V. PROTEINS AND PEPTIDES

In certain embodiments, the present invention concerns novelcompositions comprising at least one protein or peptide, such as akynureninase. These peptides may be comprised in a fusion protein orconjugated to an agent as described supra.

As used herein, a protein or peptide generally refers, but is notlimited to, a protein of greater than about 200 amino acids, up to afull length sequence translated from a gene; a polypeptide of greaterthan about 100 amino acids; and/or a peptide of from about 3 to about100 amino acids. For convenience, the terms “protein,” “polypeptide,”and “peptide” are used interchangeably herein.

As used herein, an “amino acid residue” refers to any naturallyoccurring amino acid, any amino acid derivative, or any amino acid mimicknown in the art. In certain embodiments, the residues of the protein orpeptide are sequential, without any non-amino acids interrupting thesequence of amino acid residues. In other embodiments, the sequence maycomprise one or more non-amino acid moieties. In particular embodiments,the sequence of residues of the protein or peptide may be interrupted byone or more non-amino acid moieties.

Accordingly, the term “protein or peptide” encompasses amino acidsequences comprising at least one of the 20 common amino acids found innaturally occurring proteins, or at least one modified or unusual aminoacid.

Proteins or peptides may be made by any technique known to those ofskill in the art, including the expression of proteins, polypeptides, orpeptides through standard molecular biological techniques, the isolationof proteins or peptides from natural sources, or the chemical synthesisof proteins or peptides. The nucleotide and protein, polypeptide, andpeptide sequences corresponding to various genes have been previouslydisclosed, and may be found at computerized databases known to those ofordinary skill in the art. One such database is the National Center forBiotechnology Information's Genbank and GenPept databases (available onthe world wide web at ncbi.nlm.nih.gov/). The coding regions for knowngenes may be amplified and/or expressed using the techniques disclosedherein or as would be known to those of ordinary skill in the art.Alternatively, various commercial preparations of proteins,polypeptides, and peptides are known to those of skill in the art.

VI. NUCLEIC ACIDS AND VECTORS

In certain aspects of the invention, nucleic acid sequences encoding akynureninase or a fusion protein containing a kynureninase may bedisclosed. Depending on which expression system is used, nucleic acidsequences can be selected based on conventional methods. For example, ifthe kynureninase is derived from human kynureninase and containsmultiple codons that are rarely utilized in E. coli, then that mayinterfere with expression. Therefore, the respective genes or variantsthereof may be codon optimized for E. coli expression. Various vectorsmay be also used to express the protein of interest. Exemplary vectorsinclude, but are not limited, plasmid vectors, viral vectors,transposon, or liposome-based vectors.

VII. HOST CELLS

Host cells may be any that may be transformed to allow the expressionand secretion of kynureninase and conjugates thereof. The host cells maybe bacteria, mammalian cells, yeast, or filamentous fungi. Variousbacteria include Escherichia and Bacillus. Yeasts belonging to thegenera Saccharomyces, Kiuyveromyces, Hansenula, or Pichia would find useas an appropriate host cell. Various species of filamentous fungi may beused as expression hosts, including the following genera: Aspergillus,Trichoderma, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora,Endothia, Mucor, Cochliobolus, and Pyricularia.

Examples of usable host organisms include bacteria, e.g., Escherichiacoli MC1061, derivatives of Bacillus subtilis BRB1 (Sibakov et al.,1984), Staphylococcus aureus SAI123 (Lordanescu, 1975) or Streptococcuslividans (Hopwood et al., 1985); yeasts, e.g., Saccharomyces cerevisiaeAH 22 (Mellor et al., 1983) or Schizosaccharomyces pombe; andfilamentous fungi, e.g., Aspergillus nidulans, Aspergillus awamori(Ward, 1989), or Trichoderma reesei (Penttila et al., 1987; Harkki etal., 1989).

Examples of mammalian host cells include Chinese hamster ovary cells(CHO-K1; ATCC CCL61), rat pituitary cells (GH1; ATCC CCL82), HeLa S3cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCCCRL 1548),SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650), and murineembryonic cells (NIH-3T3; ATCC CRL 1658). The foregoing beingillustrative but not limitative of the many possible host organismsknown in the art. In principle, all hosts capable of secretion can beused whether prokaryotic or eukaryotic.

Mammalian host cells expressing the kynureninase and/or their fusionproteins are cultured under conditions typically employed to culture theparental cell line. Generally, cells are cultured in a standard mediumcontaining physiological salts and nutrients, such as standard RPMI,MEM, IMEM, or DMEM, typically supplemented with 5%-10% serum, such asfetal bovine serum. Culture conditions are also standard, e.g., culturesare incubated at 37° C. in stationary or roller cultures until desiredlevels of the proteins are achieved.

VIII. PROTEIN PURIFICATION

Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the homogenization andcrude fractionation of the cells, tissue, or organ to polypeptide andnon-polypeptide fractions. The protein or polypeptide of interest may befurther purified using chromatographic and electrophoretic techniques toachieve partial or complete purification (or purification tohomogeneity) unless otherwise specified. Analytical methods particularlysuited to the preparation of a pure peptide are ion-exchangechromatography, gel exclusion chromatography, polyacrylamide gelelectrophoresis, affinity chromatography, immunoaffinity chromatography,and isoelectric focusing. A particularly efficient method of purifyingpeptides is fast-performance liquid chromatography (FPLC) or evenhigh-performance liquid chromatography (HPLC).

A purified protein or peptide is intended to refer to a composition,isolatable from other components, wherein the protein or peptide ispurified to any degree relative to its naturally-obtainable state. Anisolated or purified protein or peptide, therefore, also refers to aprotein or peptide free from the environment in which it may naturallyoccur. Generally, “purified” will refer to a protein or peptidecomposition that has been subjected to fractionation to remove variousother components, and which composition substantially retains itsexpressed biological activity. Where the term “substantially purified”is used, this designation will refer to a composition in which theprotein or peptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, or more of the proteins in the composition.

Various techniques suitable for use in protein purification are wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like, orby heat denaturation, followed by centrifugation; chromatography steps,such as ion exchange, gel filtration, reverse phase, hydroxyapatite, andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of these and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

Various methods for quantifying the degree of purification of theprotein or peptide are known to those of skill in the art in light ofthe present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity therein,assessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification, andwhether or not the expressed protein or peptide exhibits a detectableactivity.

There is no general requirement that the protein or peptide will alwaysbe provided in its most purified state. Indeed, it is contemplated thatless substantially purified products may have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

In certain embodiments a protein or peptide may be isolated or purified,for example, a kynureninase, a fusion protein containing a kynureninase,or a modified kynureninase post PEGylation. For example, a His tag or anaffinity epitope may be comprised in such a kynureninase to facilitatepurification. Affinity chromatography is a chromatographic procedurethat relies on the specific affinity between a substance to be isolatedand a molecule to which it can specifically bind. This is areceptor-ligand type of interaction. The column material is synthesizedby covalently coupling one of the binding partners to an insolublematrix. The column material is then able to specifically adsorb thesubstance from the solution. Elution occurs by changing the conditionsto those in which binding will not occur (e.g., altered pH, ionicstrength, temperature, etc.). The matrix should be a substance that doesnot adsorb molecules to any significant extent and that has a broadrange of chemical, physical, and thermal stability. The ligand should becoupled in such a way as to not affect its binding properties. Theligand should also provide relatively tight binding. It should bepossible to elute the substance without destroying the sample or theligand.

Size exclusion chromatography (SEC) is a chromatographic method in whichmolecules in solution are separated based on their size, or in moretechnical terms, their hydrodynamic volume. It is usually applied tolarge molecules or macromolecular complexes, such as proteins andindustrial polymers. Typically, when an aqueous solution is used totransport the sample through the column, the technique is known as gelfiltration chromatography, versus the name gel permeationchromatography, which is used when an organic solvent is used as amobile phase.

The underlying principle of SEC is that particles of different sizeswill elute (filter) through a stationary phase at different rates. Thisresults in the separation of a solution of particles based on size.Provided that all the particles are loaded simultaneously or nearsimultaneously, particles of the same size should elute together. Eachsize exclusion column has a range of molecular weights that can beseparated. The exclusion limit defines the molecular weight at the upperend of this range and is where molecules are too large to be trapped inthe stationary phase. The permeation limit defines the molecular weightat the lower end of the range of separation and is where molecules of asmall enough size can penetrate into the pores of the stationary phasecompletely and all molecules below this molecular mass are so small thatthey elute as a single band.

High-performance liquid chromatography (or high-pressure liquidchromatography, HPLC) is a form of column chromatography used frequentlyin biochemistry and analytical chemistry to separate, identify, andquantify compounds. HPLC utilizes a column that holds chromatographicpacking material (stationary phase), a pump that moves the mobilephase(s) through the column, and a detector that shows the retentiontimes of the molecules. Retention time varies depending on theinteractions between the stationary phase, the molecules being analyzed,and the solvent(s) used.

IX. PHARMACEUTICAL COMPOSITIONS

It is contemplated that the novel kynureninase can be administeredsystemically or locally to inhibit tumor cell growth and, mostpreferably, to kill cancer cells in cancer patients with locallyadvanced or metastatic cancers. They can be administered intravenously,intrathecally, and/or intraperitoneally. They can be administered aloneor in combination with anti-proliferative drugs. In one embodiment, theyare administered to reduce the cancer load in the patient prior tosurgery or other procedures. Alternatively, they can be administeredafter surgery to ensure that any remaining cancer (e.g., cancer that thesurgery failed to eliminate) does not survive.

It is not intended that the present invention be limited by theparticular nature of the therapeutic preparation. For example, suchcompositions can be provided in formulations together withphysiologically tolerable liquid, gel, or solid carriers, diluents, andexcipients. These therapeutic preparations can be administered tomammals for veterinary use, such as with domestic animals, and clinicaluse in humans in a manner similar to other therapeutic agents. Ingeneral, the dosage required for therapeutic efficacy will varyaccording to the type of use and mode of administration, as well as theparticularized requirements of individual subjects.

Such compositions are typically prepared as liquid solutions orsuspensions, as injectables. Suitable diluents and excipients are, forexample, water, saline, dextrose, glycerol, or the like, andcombinations thereof. In addition, if desired, the compositions maycontain minor amounts of auxiliary substances, such as wetting oremulsifying agents, stabilizing agents, or pH buffering agents.

Where clinical applications are contemplated, it may be necessary toprepare pharmaceutical compositions comprising proteins, antibodies, anddrugs in a form appropriate for the intended application. Generally,pharmaceutical compositions may comprise an effective amount of one ormore kynureninase or additional agents dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic, or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of a pharmaceutical composition thatcontains at least one kyureninase isolated by the method disclosedherein, or additional active ingredient will be known to those of skillin the art in light of the present disclosure, as exemplified byRemington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated hereinby reference. Moreover, for animal (e.g., human) administration, it willbe understood that preparations should meet sterility, pyrogenicity,general safety, and purity standards as required by the FDA Office ofBiological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed., 1990, incorporated herein by reference). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the pharmaceutical compositions is contemplated.

Certain embodiments of the present invention may comprise differenttypes of carriers depending on whether it is to be administered insolid, liquid, or aerosol form, and whether it needs to be sterile forthe route of administration, such as injection. The compositions can beadministered intravenously, intradermally, transdermally, intrathecally,intraarterially, intraperitoneally, intranasally, intravaginally,intrarectally, intramuscularly, subcutaneously, mucosally, orally,topically, locally, by inhalation (e.g., aerosol inhalation), byinjection, by infusion, by continuous infusion, by localized perfusionbathing target cells directly, via a catheter, via a lavage, in lipidcompositions (e.g., liposomes), or by other methods or any combinationof the forgoing as would be known to one of ordinary skill in the art(see, for example, Remington's Pharmaceutical Sciences, 18th Ed., 1990,incorporated herein by reference).

The modified polypeptides may be formulated into a composition in a freebase, neutral, or salt form. Pharmaceutically acceptable salts includethe acid addition salts, e.g., those formed with the free amino groupsof a proteinaceous composition, or which are formed with inorganicacids, such as, for example, hydrochloric or phosphoric acids, or suchorganic acids as acetic, oxalic, tartaric, or mandelic acid. Saltsformed with the free carboxyl groups can also be derived from inorganicbases, such as, for example, sodium, potassium, ammonium, calcium, orferric hydroxides; or such organic bases as isopropylamine,trimethylamine, histidine, or procaine. Upon formulation, solutions willbe administered in a manner compatible with the dosage formulation andin such amount as is therapeutically effective. The formulations areeasily administered in a variety of dosage forms, such as formulated forparenteral administrations, such as injectable solutions, or aerosolsfor delivery to the lungs, or formulated for alimentary administrations,such as drug release capsules and the like.

Further in accordance with certain aspects of the present invention, thecomposition suitable for administration may be provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent, or carrier is detrimental to the recipient or to thetherapeutic effectiveness of a the composition contained therein, itsuse in administrable composition for use in practicing the methods isappropriate. Examples of carriers or diluents include fats, oils, water,saline solutions, lipids, liposomes, resins, binders, fillers, and thelike, or combinations thereof. The composition may also comprise variousantioxidants to retard oxidation of one or more component. Additionally,the prevention of the action of microorganisms can be brought about bypreservatives, such as various antibacterial and antifungal agents,including but not limited to parabens (e.g., methylparabens,propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal orcombinations thereof.

In accordance with certain aspects of the present invention, thecomposition is combined with the carrier in any convenient and practicalmanner, i.e., by solution, suspension, emulsification, admixture,encapsulation, absorption, and the like. Such procedures are routine forthose skilled in the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner, such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in acomposition include buffers, amino acids, such as glycine and lysine,carbohydrates, such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle composition that includes kynureninases,one or more lipids, and an aqueous solvent. As used herein, the term“lipid” will be defined to include any of a broad range of substancesthat is characteristically insoluble in water and extractable with anorganic solvent. This broad class of compounds is well known to those ofskill in the art, and as the term “lipid” is used herein, it is notlimited to any particular structure. Examples include compounds thatcontain long-chain aliphatic hydrocarbons and their derivatives. A lipidmay be naturally occurring or synthetic (i.e., designed or produced byman). However, a lipid is usually a biological substance. Biologicallipids are well known in the art, and include for example, neutral fats,phospholipids, phosphoglycerides, steroids, terpenes, lysolipids,glycosphingolipids, glycolipids, sulphatides, lipids with ether- andester-linked fatty acids, polymerizable lipids, and combinationsthereof. Of course, compounds other than those specifically describedherein that are understood by one of skill in the art as lipids are alsoencompassed by the compositions and methods.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the kynureninase or a fusion protein thereof maybe dispersed in a solution containing a lipid, dissolved with a lipid,emulsified with a lipid, mixed with a lipid, combined with a lipid,covalently bonded to a lipid, contained as a suspension in a lipid,contained or complexed with a micelle or liposome, or otherwiseassociated with a lipid or lipid structure by any means known to thoseof ordinary skill in the art. The dispersion may or may not result inthe formation of liposomes.

The actual dosage amount of a composition administered to an animalpatient can be determined by physical and physiological factors, such asbody weight, severity of condition, the type of disease being treated,previous or concurrent therapeutic interventions, idiopathy of thepatient, and on the route of administration. Depending upon the dosageand the route of administration, the number of administrations of apreferred dosage and/or an effective amount may vary according to theresponse of the subject. The practitioner responsible for administrationwill, in any event, determine the concentration of active ingredient(s)in a composition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, an active compound may comprise between about 2% to about75% of the weight of the unit, or between about 25% to about 60%, forexample, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared in such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors, such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations, will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 milligram/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 milligram/kg/body weightto about 100 milligram/kg/body weight, about 5 microgram/kg/body weightto about 500 milligram/kg/body weight, etc., can be administered, basedon the numbers described above.

X. COMBINATION TREATMENTS

In certain embodiments, the compositions and methods of the presentembodiments involve administration of a kynureninase in combination witha second or additional therapy. Such therapy can be applied in thetreatment of any disease that is associated with kynurenine dependency.For example, the disease may be cancer.

The methods and compositions, including combination therapies, enhancethe therapeutic or protective effect, and/or increase the therapeuticeffect of another anti-cancer or anti-hyperproliferative therapy.Therapeutic and prophylactic methods and compositions can be provided ina combined amount effective to achieve the desired effect, such as thekilling of a cancer cell and/or the inhibition of cellularhyperproliferation. This process may involve administering akynureninase and a second therapy. The second therapy may or may nothave a direct cytotoxic effect. For example, the second therapy may bean agent that upregulates the immune system without having a directcytotoxic effect. A tissue, tumor, or cell can be exposed to one or morecompositions or pharmacological formulation(s) comprising one or more ofthe agents (e.g., a kynureninase or an anti-cancer agent), or byexposing the tissue, tumor, and/or cell with two or more distinctcompositions or formulations, wherein one composition provides 1) akynureninase, 2) an anti-cancer agent, or 3) both a kynureninase and ananti-cancer agent. Also, it is contemplated that such a combinationtherapy can be used in conjunction with chemotherapy, radiotherapy,surgical therapy, or immunotherapy.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic construct and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing, for example, both agents are delivered to a cellin a combined amount effective to kill the cell or prevent it fromdividing.

A kynureninase may be administered before, during, after, or in variouscombinations relative to an anti-cancer treatment. The administrationsmay be in intervals ranging from concurrently to minutes to days toweeks. In embodiments where the kynureninase is provided to a patientseparately from an anti-cancer agent, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the two compounds would still be able to exert anadvantageously combined effect on the patient. In such instances, it iscontemplated that one may provide a patient with the kynureninase andthe anti-cancer therapy within about 12 to 24 or 72 h of each other and,more particularly, within about 6-12 h of each other. In some situationsit may be desirable to extend the time period for treatmentsignificantly where several days (2, 3, 4, 5, 6, or 7) to several weeks(1, 2, 3, 4, 5, 6, 7, or 8) lapse between respective administrations.

In certain embodiments, a course of treatment will last 1-90 days ormore (this such range includes intervening days). It is contemplatedthat one agent may be given on any day of day 1 to day 90 (this suchrange includes intervening days) or any combination thereof, and anotheragent is given on any day of day 1 to day 90 (this such range includesintervening days) or any combination thereof. Within a single day(24-hour period), the patient may be given one or multipleadministrations of the agent(s). Moreover, after a course of treatment,it is contemplated that there is a period of time at which noanti-cancer treatment is administered. This time period may last 1-7days, and/or 1-5 weeks, and/or 1-12 months or more (this such rangeincludes intervening days), depending on the condition of the patient,such as their prognosis, strength, health, etc. It is expected that thetreatment cycles would be repeated as necessary.

Various combinations may be employed. For the example below akynureninase is “A” and an anti-cancer therapy is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Administration of any compound or therapy of the present embodiments toa patient will follow general protocols for the administration of suchcompounds, taking into account the toxicity, if any, of the agents.Therefore, in some embodiments there is a step of monitoring toxicitythat is attributable to combination therapy.

A. Chemotherapy

A wide variety of chemotherapeutic agents may be used in accordance withthe present embodiments. The term “chemotherapy” refers to the use ofdrugs to treat cancer. A “chemotherapeutic agent” is used to connote acompound or composition that is administered in the treatment of cancer.These agents or drugs are categorized by their mode of activity within acell, for example, whether and at what stage they affect the cell cycle.Alternatively, an agent may be characterized based on its ability todirectly cross-link DNA, to intercalate into DNA, or to inducechromosomal and mitotic aberrations by affecting nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents, such asthiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan,improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines, includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, and uracil mustard;nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, and ranimnustine; antibiotics, such as the enediyneantibiotics (e.g., calicheamicin, especially calicheamicin gammalI andcalicheamicin omegaI1); dynemicin, including dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolicacid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, and zorubicin; anti-metabolites, such asmethotrexate and 5-fluorouracil (5-FU); folic acid analogues, such asdenopterin, pteropterin, and trimetrexate; purine analogs, such asfludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidineanalogs, such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine;androgens, such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, and testolactone; anti-adrenals, such as mitotane andtrilostane; folic acid replenisher, such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpolysaccharidecomplex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g.,paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine;platinum coordination complexes, such as cisplatin, oxaliplatin, andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitoxantrone; vincristine; vinorelbine; novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan(e.g., CPT-11); topoisomerase inhibitor RFS 2000;difluorometlhylornithine (DMFO); retinoids, such as retinoic acid;capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien,navelbine, farnesyl-protein transferase inhibitors, transplatinum, andpharmaceutically acceptable salts, acids, or derivatives of any of theabove.

B. Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated, such as microwaves, proton beamirradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), andUV-irradiation. It is most likely that all of these factors affect abroad range of damage on DNA, on the precursors of DNA, on thereplication and repair of DNA, and on the assembly and maintenance ofchromosomes. Dosage ranges for X-rays range from daily doses of 50 to200 roentgens for prolonged periods of time (3 to 4 wk), to single dosesof 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely,and depend on the half-life of the isotope, the strength and type ofradiation emitted, and the uptake by the neoplastic cells.

C. Immunotherapy

The skilled artisan will understand that immunotherapies may be used incombination or in conjunction with methods of the embodiments. In thecontext of cancer treatment, immunotherapeutics, generally, rely on theuse of immune effector cells and molecules to target and destroy cancercells. Rituximab (RITUXAN®) is such an example. Checkpoint inhibitors,such as, for example, ipilumimab, are another such example. The immuneeffector may be, for example, an antibody specific for some marker onthe surface of a tumor cell. The antibody alone may serve as an effectorof therapy or it may recruit other cells to actually affect cellkilling. The antibody also may be conjugated to a drug or toxin(chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussistoxin, etc.) and serve merely as a targeting agent. Alternatively, theeffector may be a lymphocyte carrying a surface molecule that interacts,either directly or indirectly, with a tumor cell target. Variouseffector cells include cytotoxic T cells and NK cells.

In one aspect of immunotherapy, the tumor cell must bear some markerthat is amenable to targeting, i.e., is not present on the majority ofother cells. Many tumor markers exist and any of these may be suitablefor targeting in the context of the present embodiments. Common tumormarkers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68,TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor,erb B, and p155. An alternative aspect of immunotherapy is to combineanticancer effects with immune stimulatory effects. Immune stimulatingmolecules also exist including: cytokines, such as IL-2, IL-4, IL-12,GM-CSF, gamma-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growthfactors, such as FLT3 ligand.

Examples of immunotherapies currently under investigation or in use areimmune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum,dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998);cytokine therapy, e.g., interferons α, β, and γ, IL-1, GM-CSF, and TNF(Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998);gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998;Austin-Ward and Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and5,846,945); and monoclonal antibodies, e.g., anti-CD20, anti-gangliosideGM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Pat.No. 5,824,311). It is contemplated that one or more anti-cancertherapies may be employed with the antibody therapies described herein.

D. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative, andpalliative surgery. Curative surgery includes resection in which all orpart of cancerous tissue is physically removed, excised, and/ordestroyed and may be used in conjunction with other therapies, such asthe treatment of the present embodiments, chemotherapy, radiotherapy,hormonal therapy, gene therapy, immunotherapy, and/or alternativetherapies. Tumor resection refers to physical removal of at least partof a tumor. In addition to tumor resection, treatment by surgeryincludes laser surgery, cryosurgery, electrosurgery, andmicroscopically-controlled surgery (Mohs' surgery).

Upon excision of part or all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection, or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

E. Other Agents

It is contemplated that other agents may be used in combination withcertain aspects of the present embodiments to improve the therapeuticefficacy of treatment. These additional agents include agents thataffect the upregulation of cell surface receptors and GAP junctions,cytostatic and differentiation agents, inhibitors of cell adhesion,agents that increase the sensitivity of the hyperproliferative cells toapoptotic inducers, or other biological agents. Increases inintercellular signaling by elevating the number of GAP junctions wouldincrease the anti-hyperproliferative effects on the neighboringhyperproliferative cell population. In other embodiments, cytostatic ordifferentiation agents can be used in combination with certain aspectsof the present embodiments to improve the anti-hyperproliferativeefficacy of the treatments. Inhibitors of cell adhesion are contemplatedto improve the efficacy of the present embodiments. Examples of celladhesion inhibitors are focal adhesion kinase (FAKs) inhibitors andLovastatin. It is further contemplated that other agents that increasethe sensitivity of a hyperproliferative cell to apoptosis, such as theantibody c225, could be used in combination with certain aspects of thepresent embodiments to improve the treatment efficacy.

XI. KITS

Certain aspects of the present invention may provide kits, such astherapeutic kits. For example, a kit may comprise one or morepharmaceutical composition as described herein and optionallyinstructions for their use. Kits may also comprise one or more devicesfor accomplishing administration of such compositions. For example, asubject kit may comprise a pharmaceutical composition and catheter foraccomplishing direct intravenous injection of the composition into acancerous tumor. In other embodiments, a subject kit may comprisepre-filled ampoules of a kynureninase, optionally formulated as apharmaceutical, or lyophilized, for use with a delivery device.

Kits may comprise a container with a label. Suitable containers include,for example, bottles, vials, and test tubes. The containers may beformed from a variety of materials, such as glass or plastic. Thecontainer may hold a composition that includes a kynureninase that iseffective for therapeutic or non-therapeutic applications, such asdescribed above. The label on the container may indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, and may also indicate directions for either in vivo or invitro use, such as those described above. The kit of the invention willtypically comprise the container described above and one or more othercontainers comprising materials desirable from a commercial and userstandpoint, including buffers, diluents, filters, needles, syringes, andpackage inserts with instructions for use.

XII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1—Gene Construction, Expression, and Purification ofKynureninase from Psuedomonas fluorescens

A gene for expression of the kynureninase enzyme from Pseudomonasfluorescens (Pf-KYNU) was constructed by overlap extension polymerasechain reaction (PCR) of four codon optimized gene blocks designed usingDNA-Works software (Hoover and Lubkowski, 2002). The full-length geneincludes an N-terminal XbaI restriction enzyme site (nucleotides 1-6),an optimized ribosome binding site (RBS; nucleotides 29-55), a startcodon (nucleotides 56-58), an N-terminal His₆ tag (nucleotides 59-91),an E. coli codon optimized Pf-KYNU gene (nucleotides 92-1336), a stopcodon (nucleotides 1337-1342), and a C-terminal BamHI restriction enzymesite (nucleotides 1342-1347) (see, SEQ ID NO: 1). The aforementionedrestriction enzyme sites were used to clone the assembled gene into apET-28a+ vector (Novagen). This construct was then used to transformBL21 (DE3) E. coli for expression. Cells were grown at 37° C. withshaking at 210 rpm in Terrific Broth (TB) media with 50 mg/L ofkanamycin. Expression was induced when an OD₆₀₀˜1.0 was reached byadding IPTG (0.5 mM final concentration) with continued shakingovernight at 37° C. Cells were then harvested by centrifugation andre-suspended in lysis buffer consisting of 50 mM sodium phosphate, pH7.4, 300 mM NaCl, 0.5 mM pyridoxyl phosphate (PLP), 1 mMphenylmethylsulfonylfluoride, and 1 μg/mL DNase. Lysis was achieved byFrench press and the lysate was cleared of particulates by centrifugingat 20,000×g for 1 h at 4° C. The supernatant was then filtered through a5 μm syringe filter and applied to a Ni-NTA/agarose column (Qiagen)pre-equilibrated in a buffer composed of 50 mM sodium phosphate, 300 mMNaCl, and 0.1 mM PLP at pH 7.4. After loading the lysate onto thecolumn, the resin was washed with 5 column volumes (CV) of 50 mM sodiumphosphate, pH 7.4, 300 mM NaCl, and 0.1 mM PLP with 30 mM imidazole.Next, the flow rate was set to slowly wash the column overnight with 100CV of endotoxin-free PBS (Corning) buffer with 0.1 mM PLP and 1% v/vTRITON® X114. This overnight wash removes lipopolysaccharide (LPS orendotoxin) that is a typical contaminant of bacterial expressionsystems. The washed enzyme was then eluted in 5 CV of endotoxin-free PBSwith 0.1 mM PLP with 250 mM imidazole, and the resin was rinsed with asecond 5 CV portion of endotoxin free PBS with 0.1 mM PLP. At thispoint, enzyme was buffer exchanged into fresh PBS to remove imidazole,10% glycerol was added and aliquots were flash frozen in liquid nitrogenfor storage at −80° C. Alternatively, enzyme was immediately bufferexchanged into freshly made, sterile 100 mM sodium phosphate, pH 8.4, toboth remove imidazole and prepare it for PEGylation (see, Example 4).Enzyme purities were typically >95% based on SDS-PAGE analysis andtypical yields averaged around 75 mg/L of culture. Protein quantitieswere assessed by measuring Abs_(280 nm) and using the calculated enzymeextinction coefficient of 63,745 M⁻¹cm⁻¹.

Example 2—Gene Construction, Expression, and Purification ofKynureninase from Homo sapiens

A gene for expression of the kynureninase enzyme from Homo sapiens(h-KYNU) was obtained by overlap extension polymerase chain reaction(PCR) of four codon optimized gene blocks designed using DNA-Workssoftware (Hoover and Lubkowski, 2002). The full-length gene includes anN-terminal XbaI restriction enzyme site (nucleotides 1-6), an optimizedRBS (nucleotides 28-60), a start codon (nucleotides 61-63), anN-terminal His₆ tag (nucleotides 64-96), an E. coli codon optimizedh-KYNU gene (nucleotides 97-1488), a stop codon (nucleotides 1489-1491),and a C-terminal BamHI restriction enzyme site (nucleotides 1492-1497)(see, SEQ ID NO: 2). The aforementioned restriction enzyme sites wereused to clone the assembled gene into a pET-28a+ vector (Novagen). Thisconstruct was then used to transform BL21 (DE3) E. coli for expression.Cells were grown at 37° C. with shaking at 210 rpm in Terrific Broth(TB) media with 50 mg/L of kanamycin. Expression was induced when anOD₆₀₀˜1.0 was reached by adding IPTG (0.5 mM final concentration) withcontinued shaking overnight at 37° C. Cells were then harvested bycentrifugation and re-suspended in lysis buffer consisting of 50 mMsodium phosphate, pH 7.4, 300 mM NaCl, 0.5 mM pyridoxyl phosphate (PLP),1 mM phenylmethylsulfonylfluoride, and 1 μg/mL DNase. Lysis was achievedby French press and the lysate was cleared of particulates bycentrifuging at 20,000×g for 1 h at 4° C. The supernatant was thenfiltered through a 5 μm syringe filter and applied to a Ni-NTA/agarosecolumn (Qiagen) pre-equilibrated in 50 mM sodium phosphate, pH 7.4, 300mM NaCl, and 0.1 mM PLP buffer. After loading the lysate onto thecolumn, the resin was washed with 5 column volumes (CV) of 50 mM sodiumphosphate, pH 7.4, 300 mM NaCl, and 0.1 mM PLP with 30 mM imidazole.Next, the flow rate was set to slowly wash the column overnight with 100CV of endotoxin-free PBS (Corning) buffer with 0.1 mM PLP and 1% v/vTRITON® X114. This overnight wash removes lipopolysaccharide (LPS orendotoxin) that is a typical contaminant in bacterial expression ofenzymes. The washed enzyme was then eluted in 5 CV of endotoxin free PBSwith 0.1 mM PLP with 250 mM imidazole and the resin was rinsed with asecond 5 CV portions of endotoxin free PBS with 0.1 mM PLP. At thispoint, enzyme was buffer exchanged into fresh PBS to remove imidazole,10% glycerol was added and aliquots were flash frozen in liquid nitrogenfor storage at −80° C. Alternatively, enzyme could be buffer exchangedinto freshly made, sterile 100 mM sodium phosphate, pH 8.4, to bothremove imidazole and prepare it for PEGylation (see, Example 4). Enzymepurities were typically >95% as assessed by SDS-PAGE analysis andtypical yields averaged around 20 mg/L of liquid culture. Proteinquantities were assessed by measuring Abs_(280 nm) and using thecalculated enzyme extinction coefficient of 76,040 M⁻¹cm⁻¹.

Example 3—Gene Construction, Expression, and Purification ofKynureninase from Mus musculus

A gene for expression of the kynureninase enzyme from Mus musculus(m-KYNU) was obtained by overlap extension polymerase chain reaction(PCR) of three codon optimized gene blocks designed using DNA-Workssoftware (Hoover et al., 2002). The full-length gene included anN-terminal XbaI restriction enzyme site (nucleotides 1-6), an optimizedRBS (nucleotides 29-58), a start codon (nucleotides 59-61), anN-terminal His₆ tag (nucleotides 62-94), an E. coli codon optimizedm-KYNU gene (nucleotides 95-1483), a stop codon (nucleotides 1484-1486),and a C-terminal BamHI restriction enzyme site (nucleotides 1487-1492)(see, SEQ ID NO: 3). The aforementioned restriction enzyme sites wereused to clone the assembled gene into a pET-28a+ vector (Novagen). Thisconstruct was then used to transform BL21 (DE3) E. coli for expression.Cells were grown at 37° C. shaking at 210 rpm in Terrific Broth (TB)media with 50 mg/L of kanamycin. Expression was induced when anOD₆₀₀˜1.0 was reached by adding 0.5 mM IPTG and continued overnight at37° C. Cells were harvested by centrifugation and re-suspended in lysisbuffer consisting of 50 mM sodium phosphate, pH 7.4, 300 mM NaCl, 0.5 mMpyridoxyl phosphate (PLP), 1 mM phenylmethylsulfonylfluoride, and 1μg/mL DNase. Lysis was achieved by French press and the lysate clearedof particulates by centrifuging at 20,000×g for 1 h at 4° C. Thesupernatant was filtered through a 5 μm syringe filter and applied to aNi-NTA/agarose column (Qiagen) pre-equilibrated in 50 mM sodiumphosphate, pH 7.4, 300 mM NaCl, and 0.1 mM PLP buffer. After loading thelysate onto the column, the resin was washed with 5 column volumes (CV)of 50 mM sodium phosphate, pH 7.4, 300 mM NaCl, and 0.1 mM PLP with 30mM imidazole. Next the flow rate was set to slowly wash overnight with100 CV of endotoxin-free PBS (Corning) buffer with 0.1 mM PLP and 1% v/vTRITON® X114. This overnight wash removeD lipopolysaccharide (LPS orendotoxin) that is a typical contaminant in bacterial expression ofenzymes. The washed enzyme was eluted in 5 CV of endotoxin-free PBS with0.1 mM PLP with 250 mM imidazole and the resin rinsed with a second 5 CVportion of endotoxin-free PBS with 0.1 mM PLP. At this point, enzyme wasbuffer exchanged into fresh PBS to remove imidazole, 10% glycerol addedand aliquots flash frozen in liquid nitrogen for storage at −80° C.

Example 4—Pharmacological Preparation of Kynureninase from Pseudomonasfluorescens

To improve the circulation time of the enzyme in vivo, the hydrodynamicradius of KYNU enzymes was increased by functionalizing surface reactivegroups in the protein by conjugation to PEG. In one embodiment, Pf-KYNUwas functionalized by reaction of surface lysine residues with MethoxylPEG Succinimidyl Carbonate 5000 MW (NANOCS). The purified,endotoxin-free enzyme was thoroughly buffer exchanged into freshlyprepared 100 mM sodium phosphate, pH 8.4, and concentrated to 10 mg/mL.The resulting solution was added directly to a 100:1 molar excess ofsolid PEG reagent and allowed to react at room temperature for 1 h (FIG.1). Un-reacted PEG was removed from solution by thorough buffer exchangeinto fresh, endotoxin-free PBS in a 100 kDa cut off centrifugalfiltration device (AMICON®). The apparent molecular mass of the enzymewas then checked on a size exclusion HPLC column (Phenomenex) in PBS. AMW standard solution from BioRad was used to generate a standard curveand enzyme retention times compared to those of the protein standards.Based on the standard curve, the non-PEGylated enzyme has an apparentmass of 40 kDa, which is close to that of the mass of one monomer ofPf-KYNU. The PEGylated version of the enzyme was seen to have anapparent mass of 1,300 kDa, i.e. substantially larger than theunmodified enzyme. Endotoxin levels were quantified using the Chromo-LALkinetic chromogenic endotoxin testing kit (Associates of Cape Cod,Inc.). Enzyme washed in the manner described above typically resulted inendotoxin levels 0.19±0.07 EU/mg of purified Pf-KYNU.

Example 5—Pharmacological Preparation of Kynureninase from Homo sapiens

To improve circulatory residence time of the human enzyme in vivo, thehydrodynamic radius of h-KYNU was increased by functionalizing surfacereactive groups in the protein by conjugation to PEG. In one embodiment,h-KYNU was functionalized by reaction of surface lysine residues withMethoxyl PEG Succinimidyl Carbonate 5000 MW (NANOCS). The purified,endotoxin-free enzyme was thoroughly buffer exchanged into freshlyprepared 100 mM sodium phosphate, pH 8.4, and concentrated to 10 mg/mL.The resulting solution was added directly to a 100:1 molar excess ofsolid PEG reagent and allowed to react at room temperature for 1 h.Un-reacted PEG was removed from solution by thorough buffer exchangeinto fresh, endotoxin-free PBS in a 100 kDa cut off centrifugalfiltration device (AMICON®). The apparent molecular mass of the enzymewas determined using a size exclusion HPLC column (Phenomenex)equilibrated with PBS and retention times compared to a MW standardsolution (BioRad). Endotoxin levels were quantified using the Chromo-LALkinetic chromogenic endotoxin testing kit (Associates of Cape Cod,Inc.).

Example 6—Assay for Measuring Kinetic Parameters of Kynureninase

The kinetic parameters of Pf-KYNU and h-KYNU, as well as of theirPEGylated versions as described in Examples 4 and 5, were quantified bya spectrophotometric assay, in which the decay in the maximum absorbanceof the enzyme substrate, L-kynurenine, was monitored as a function oftime. L-kynurenine solutions were prepared in a PBS buffer, pH 7.4, toresult in final concentrations ranging from 8 μM to 250 μM. L-Kynureninehas an extinction coefficient of 4,500 M⁻¹cm⁻¹ with a λ_(max) at 365 nmwhile the products of the kynureninase reaction, L-anthranilic acid andL-alanine, do not appreciably absorb at 365 nm. Reactions were initiatedby adding and rapidly mixing enzyme solutions (˜20 nM final) with thesubstrate solutions and monitoring the loss of substrate KYN at 25° C.by measuring Abs_(365 nm) over time. The resulting data was processedand fitted to the Michaelis-Menten equation for determining kineticconstants. The kinetics of PEGylated Pf-KYNU enzyme was measured in anidentical manner. For the non-PEGylated enzyme, k_(cat)/K_(M)=1.0×10⁵M⁻¹s⁻¹, and for the PEGylated form, k_(cat)/K_(M)=1.3×10⁵ M⁻¹s⁻¹.Kinetic parameters for the hydrolysis of 3-hydroxy-L-kynurenic acid werealso determined as described here.

Example 7—In Vitro Stability of Kynureninase

To measure the in vitro stability of Pf-KYNU, the enzyme was added toeither PBS buffer or pooled human serum to a final concentration of 10μM and incubated at 37° C. Portions of 10 μL each were taken out at timepoints and added to 990 μL of a 250 μM solution of L-kynurenine/PBS. Theinitial rate of reaction was monitored by measuring the decay ofabsorbance at 365 nm over time as described in Example 3. Enzymestability was determined by comparing the initial rate of L-kynureninecatalysis at each time point and comparing it to the rate at time=0. Theresulting data was plotted as % activity vs. time and fitted to anexponential equation to determine the half-life (T_(1/2)). The Pf-KYNUenzyme was found to have a T_(1/2)=34.3 hours in PBS and a T_(1/2)=2.4hours in pooled human serum (FIG. 2).

Example 8—Assay for Quantifying Kynurenine and Tryptophan Levels In Vivo

In vivo levels of L-kynurenine, tryptophan, kynureninic acid,3-hydroxy-L-kynurenine and L-anthranlilic acid (one of the products ofkynureninase catalysis) were quantified and monitored by HPLC. Uponnecropsy of the mice, samples of blood, the tumor, the spleen, and theliver were removed. Blood samples were centrifuged to separate wholeblood from serum. Tissue samples were first homogenized, and thencentrifuged to remove the solid portion. To each liquid portion wasadded a 1:10 v/v portion of 100% trichloroacetic acid to precipitatemacromolecules. Solids were again removed by centrifuging and thesupernatants were passed through a 0.45 μm syringe filter. The treatedsupernatants were applied directly to a HPLC (Shimadzu) and separated ona standard analytical C-18 column using a gradient starting from 0%solution B to 100% solution B where solution A is H₂O+0.1%trifluoroacetic acid and solution B is acetonitrile+0.1% trifluoroaceticacid. The full absorbance range from 190 nm to 900 nm was continuallycollected to monitor all possible molecules and fluorescencespectroscopy (Ex=365 nm, Em=480 nm) was simultaneously collected tospecifically monitor kynurenine levels. Concentrations and retentiontimes were determined using standard solutions made from the puremolecules (Sigma).

Example 9—Efficacy of PEG-Pf-KYNU in the Autologous B16 Mouse MelanomaModel

B6-WT mice (n=20) were each inoculated with 2.5×10⁵ B16 murine melanomacells by subcutaneous flank injection. After allowing tumors toestablish for 10 days (tumor mean=20 mm²) the mice were split into twogroups of n=10 each. The control group was then treated with 20 mg/kg ofheat inactivated PEG-Pf-KYNU by intra-tumoral injection every three daysuntil tumors reached 350 mm² in size. The experimental group was treatedin an identical manner except with 20 mg/kg of active PEG-Pf-KYNU byintra-tumoral injection every three days until tumors reached anendpoint of 350 mm² in size. The growth rates of B16 melanoma tumors wassignificantly retarded in the treatment group administered activePEG-Pf-KYNU compared to the identically treated heat-inactivatedPEG-Pf-KYNU group (FIG. 3) resulting in a significant life-spanextension (FIG. 4). Lymphocytes isolated from control and experimentaltreatment groups were assessed with panels of antibodies (i.e.,anti-CD45, CD4, Nk1.1, CD25, FoxP3, CD8, granzyme B, IFNγ, CTLA4, CD11c,CD11b, F4/80, GR-1, and Ly6-C) which revealed that the population ofcirculating CD4+ CD25+ FoxP3+ regulatory T-cells was significantly lowerin the group treated with active PEG-Pf-KYNU (4.8±0.8% vs. 8.6±0.8%). Inaddition, the population of tumor infiltrating CD8+ T-cells expressinggranzyme B and interferon γ was significantly higher in mice treatedwith active enzyme (26±19% vs. 4±2%) (FIGS. 5A-B).

Example 10—Kynureninase-scFv Fusion Proteins for Tumor Targeting

In some aspects, the present invention also contemplates polypeptidescomprising the modified bacterial or mammalian kynureninase linked to aheterologous amino acid sequence. For example, the native or modifiedkynureninase may be linked to a single-chain variable fragment (scFv)antibody that binds specific cell surface tumor antigens. In thisembodiment an scFv-kynureninase fusion protein with the scFv portion ofthe protein having specific affinity for a known tumor antigen,preferably a tumor specific antigen that internalizes at a slower rate,e.g., MUC-1, would allow the kynureninase portion of the fusion proteinto be delivered to the tumor cell and degrade KYN. One example would bea scFv-kynureninase fusion protein where the scFv portion targets andbinds to the human epidermal growth factor receptor 2 (HER2) that isupregulated in certain types of breast cancer.

In this embodiment a native or modified kynureninase-anti-HER2-scFVfusion protein would act to target and concentrate kynureninase directlyto the tumor surface and act to degrade tumor-produced KYN.

Example 11—Kynureninase-Anti-CTLA4-scFv Fusion Proteins

In some aspects, the present invention also contemplates polypeptidescomprising the modified bacterial or mammalian kynureninase linked to aheterologous amino acid sequence. For example, the native or modifiedkynureninase may be linked to a single-chain variable fragment (scFv)antibody that binds the Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4)receptor, Programmed Cell Death 1 (PD-1), or Programmed Cell DeathLigand 1 (PD-L1). A blockade of CTLA-4, PD-1, or PD-L1 by anantagonizing antibody or antibody fragment allows the inhibitory T-cellsignal to be reversed allowing CD28 to stimulate T-cell activation. Inthis embodiment a native or modified kynureninase-anti-CTLA4-,anti-PD-1-, or anti-PD-L1-scFv fusion protein would act to remove bothinhibitory protein:protein interaction signaling and inhibitorykynurenine signaling. This embodiment of a native or modifiedkynureninase-scFv fusion protein would be expected to potentlyupregulate T-cell activation and promote robust anti-tumoral responses.

Example 12—Chimeric Antigen Receptor Constructs for Delivery ofKynureninase to T Cells

In some aspects, the present invention also contemplates a lentiviralvector suitable for transfection of T cells with chimeric antigenreceptor (CAR) constructs such that a modified bacterial or mammaliankynureninase would be co-expressed in addition to the CAR construct. CARconstructs are proteins containing an extracellular antigen bindingdomain fused to a transmembrane and cytoplasmic signaling domain from aCD3-ζ chain and often a CD28 molecule (Ahmed et al., 2010). The antigenbinding domain may be an scFv designed to bind an antigen expressed by atumor cell with examples being HER2 expressed by glioblastoma orosteosarcoma, CD19 or CD20 expressed by various B-cell malignancies, orGD2 expressed by neuroblastoma (Lipowska-Bhalla et al., 2012) or anyother relevant target. In this embodiment the lentiviral vector,delivering an appropriate CAR construct to a T cell, would in additionco-express a native or modified bacterial or mammalian kynureninase inthe cytosol. The T cell containing this CAR/kynureninase construct wouldhave the dual ability to 1) bind to specific tumor cells and 2) todegrade KYN, preventing KYN induction of a regulatory phenotype and orapoptosis. In another embodiment a T cell would express a CAR constructthat binds a CD19+ or CD20+ diffuse large B-cell lymphoma whileco-expressing a kynureninase to degrade the high concentrations of KYNoften produced by this tumor type (Yoshikawa et al., 2010; de Jong etal., 2011; Yao et al., 2011).

Example 13—Genetic Selection for Kynureninase Activity

The amino acid L-tryptophan (L-Trp) is synthesized from the pentosederived precursor, chorismate, by expression of the trp biosyntheticgenes. In bacteria such as E. coli the trp biosynthetic genes areorganized in an operon composed of five genes; trpE, trpD, trpC, trpB,and trpA. The TrpE and TrpD proteins are components of the anthranilatesynthase complex that catalyzes the first step in the conversion ofchorismate and L-glutamine to anthranilic acid and L-glutamate.Anthranilic acid is then subsequently converted to L-Trp by the actionof TrpC, TrpA, and TrpB. Cells lacking a functional anthranilatesynthase gene are auxotrophic for L-Trp and cannot grow in minimal mediawithout tryptophan. The inventors postulated that since kynurenine canbe transported into the cytosol of many organisms, cells expressingrecombinant L-kynureninase enzymes displaying a sufficiently highcatalytic activity should be able to convert cytosolic L-kynurenine toanthranilic acid and the latter then enables the synthesis of L-Trp. Bycontrast, cells that do not express the enzyme or express variants withlow catalytic activity should display either no growth or very slowgrowth, respectively, on minimal media with L-kynurenine.

E. coli trpE and trpD deletion mutants were obtained from GeneticResources at Yale CGSC. Strain genotypes were (F-, Δ(araD-araB)567,AlacZ4787(::rrnB-3), λ-, ΔtrpE772::kan, rph-1, Δ(rhaD-rhaB)568, hsdR514)and (F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ-, λtrpD771::kan, rph-1,Δ(rhaD-rhaB)568, hsdR514), respectively. Cells were plated on M9 minimalmedia plates. Filter paper disks soaked in either L-Trp, L-Kyn,anthranilic acid, or buffer were then placed on the plates followed byincubation at 37° C. E. coli-ΔtrpD cells only grew in the presence ofL-Trp, however E. coli-ΔtrpE could also grow in the presence ofanthranilic acid but not buffer or L-Kyn, demonstrating that trpC, trpA,and trpB were expressed, allowing rescue of the L-Trp auxotrophy withanthranilic acid as an intermediate metabolite (FIG. 6). Furthermore, E.coli-ΔtrpE cells transformed with a plasmid harboring the Pf-KYNU genegrew robustly on M9 minimal media plates in the presence of L-Kyn.

Example 14—Gene Construction, Expression and Purification of BacterialKynureninases Displaying High Catalytic Activity Towards Kynurenine andIdentity to the Human Kynureninase

Similar to other eukaryotic kynureninases the Homo sapiens enzyme ishighly selective towards the hydrolysis of 3′-OH kynurenine and hasabout 1,000-fold lower catalytic activity towards kynurenine. Because ofits poor catalytic activity towards kynurenine, the human enzyme is notsuitable for therapeutic purposes. Administration of PEGylated Pf-KYNU(Example 9), Mu-KYNU (Example 22 and Example 23), or Cp-KYNU (Example17) (all of which display high catalytic activity towards kynurenineinstead of 3′-OH kynurenine) resulted in tumor growth retardation asshown in Example 9 (FIG. 3). However, administration of PEGylated humankynureninase at similar or higher dosing had no effect on the growth ofB16 melanoma tumors (n=4). However, as shown in Example 20, engineeringof h-KYNU can improve the L-kynurenine degrading activity of the humanenzyme. Such engineered h-KYNU variants may result in tumor growthretardation as seen with PEGylated Pf-KYNU (Example 9), Mu-KYNU (Example22 and Example 23), and Cp-KYNU (Example 17).

The Pf-KYNU displays low sequence identity to its human counterpart (24%amino acid identity). Due to its low sequence identity to the humanprotein, Pf-KYNU may elicit adverse immune responses in patients as wellas the production of neutralizing antibodies. Therefore it is importantto discover kynureninase enzymes that display high catalytic activityand selectivity towards kynurenine and have a higher degree of aminoacid identity to the Homo sapiens kynureninase. The inventors identifieda number of bacterial enzymes that display >38% amino acid identity tothe Homo sapiens kynureninase and also high kynurenine hydrolysisactivity. The sequences of these enzymes are provided as SEQ ID NOs:13-52. The percent identities of these enzymes as compared to Homosapiens kynureninase are provided in Table 1. As a representativeexample, a gene for expression of the kynureninase enzyme fromMucilaginibacter paludis (Mu-KYNU) (SEQ ID NO: 33) was constructed byoverlap extension polymerase chain reaction (PCR) of two codon optimizedgene blocks designed using the DNA-Works software (Hoover and Lubkowski,2002). The full-length gene includes an N-terminal NcoI restrictionenzyme site, an optimized RBS, an N-terminal His₆ tag, E. coli codonoptimized Mu-KYNU gene, a stop codon and a C-terminal EcoRI restrictionenzyme site. The aforementioned restriction enzyme sites were used toclone the assembled gene into a pET-28a+ vector (Novagen). Thisconstruct was then used to transform BL21 (DE3) E. coli for expression.Cells were grown at 37° C. with shaking at 210 rpm in Terrific Broth(TB) media with 50 mg/L of kanamycin. Expression was induced when anOD₆₀₀˜1.0 was reached by adding IPTG (0.5 mM final concentration) withcontinued shaking overnight at 37° C. Cells were then harvested bycentrifugation and re-suspended in lysis buffer consisting of 50 mMsodium phosphate, pH 7.4, 300 mM NaCl, 0.5 mM pyridoxyl phosphate (PLP),1 mM phenylmethylsulfonylfluoride, and 1 μg/mL DNase. Lysis was achievedby French press and the lysate was cleared of particulates bycentrifuging at 20,000×g for 1 h at 4° C. The supernatant was thenfiltered through a 5 μm syringe filter and applied to a Ni-NTA/agarosecolumn (Qiagen) pre-equilibrated in 50 mM sodium phosphate, pH 7.4, 300mM NaCl, and 0.1 mM PLP buffer. After loading the lysate onto thecolumn, the resin was washed with 5 column volumes (CV) of 50 mM sodiumphosphate, pH 7.4, 300 mM NaCl, and 0.1 mM PLP with 30 mM imidazole. Thewashed enzyme was then eluted in 5 CV of PBS with 0.1 mM PLP with 250 mMimidazole. At this point, enzyme was buffer exchanged into fresh PBS toremove imidazole, 10% glycerol was added and aliquots were flash frozenin liquid nitrogen for storage at −80° C. Enzyme purities weretypically >95% based on SDS-PAGE analysis and typical yields averagedaround 75 mg/L of culture. Protein quantities were assessed by measuringAbs_(280 nm) and using the calculated enzyme extinction coefficient of78,185 M⁻¹cm⁻¹.

TABLE 1 Percent identities of eubacterial kynureninase enzymes ascompared to Homo sapiens kynureninase. Species SEQ ID NO % IdentityArenitalea lutea 13 44.1 Belliella baltica DSM 15883 14 43.3 Bizioniaargentinensis 15 42.9 Candidatus Entotheonella sp. TSY2 16 44.9Candidatus Koribacter versatilis Ellin345 17 43.3 Cecembia lonarensis 1845.1 Chlamydia pecorum PV3056/3 19 38.2 Chlamydophila caviae GPIC 2040.8 Corallococcus coralloides DSM 2259 21 43 Cyclobacterium marinum DSM74 22 44.5 Cystobacter fuscus 23 43.5 Echinicola vietnamensis DSM 1752624 44.5 Flavobacteria bacterium BBFL7 25 43.4 Flexibacter litoralis DSM6794 26 47.5 Formosa sp. AK20 27 45.7 Fulvivirga imtechensis 28 47.1Kangiella aquimarina 29 44.1 Kangiella koreensis DSM 16069 30 44.3Lacinutrix sp. 5H-3-7-4 31 44.2 Mariniradius saccharolyticus 32 44.5Mucilaginibacter paludis 33 43.9 Myroides odoratimimus 34 42.2Myxococcus fulvus HW-1 35 44.5 Myxococcus stipitatus DSM 14675 36 44.4Myxococcus xanthus DK 1622 37 45.1 Nafulsella turpanensis 38 48.2Niastella koreensis GR20-10 39 44.8 Nonlabens dokdonensis DSW-6 40 44Pedobacter agri 41 44.1 Pedobacter sp. BAL39 42 42.1 Pedobacter sp. V4843 44.1 Rhodonellum psychrophilum 44 45.4 Salinispora arenicola 45 39.1Saprospira grandis str. Lewin 46 43.2 Stigmatella aurantiaca DW4/3-1 4742.5 Xanthomonas axonopodis 48 42 Psychroflexus gondwanensis 49 44Lewinella cohaerens 50 45.6 Lewinella persica 51 44.9 Pontibacter roseus52 44.8

Example 15—Kinetic Parameters of Mucilaginibacter paludis Kynureninase(Mu-KYNU)

The kinetic parameters of Mu-KYNU were quantified by aspectrophotometric assay, in which the decay in the maximum absorbanceof the enzyme substrate, L-kynurenine, was monitored as a function oftime. L-Kynurenine solutions were prepared in a PBS buffer, pH 7.4, toresult in final concentrations ranging from 16 μM to 500 μM.L-Kynurenine has an extinction coefficient of 4,500 M⁻¹cm⁻¹ with aλ_(max) at 365 nm while the products of the kynureninase reaction,L-anthranilic acid and L-alanine, do not appreciably absorb at 365 nm.Reactions were initiated by adding and rapidly mixing enzyme solutions(˜20 nM final concentration) with the substrate solutions and monitoringthe loss of substrate at 25° C. by measuring Abs_(365 nm) over time. Theresulting data were processed and fitted to the Michaelis-Mentenequation for determining kinetic constants. Mu-KYNU was determined tohave a k_(cat)/K_(M)=1.2×10⁵ M⁻¹s⁻¹.

Example 16—In Vitro Stability of Mucilaginibacter paludis Kynureninase(Mu-KYNU)

To measure the in vitro stability of Mu-KYNU, the enzyme was added toeither PBS buffer or pooled human serum to a final concentration of 10μM and incubated at 37° C. Portions of 10 μL each were taken out at timepoints and added to 990 μL of a 250 μM solution of L-kynurenine/PBS. Theinitial rate of reaction was monitored by measuring the decay ofabsorbance at 365 nm over time as described in Example 3. Enzymestability was determined by comparing the initial rate of L-kynureninecatalysis at each time point and comparing it to the rate at time=0. Theresulting data were plotted as percent activity vs. time and fitted to abi-phasic decay model (Stone et al., 2010) to determine the half-lives(T_(1/2)). The activity of Mu-KYNU enzyme in PBS was found have a¹T_(1/2)=6 h with an amplitude of 74% remaining activity and asubsequent ²T_(1/2)=150 h (FIG. 7). The stability of Mu-KYNU enzyme inpooled human serum was determined to have a ¹T_(1/2)=5 h with anamplitude of 30% remaining activity and a subsequent ²T_(1/2)=73 h (FIG.7).

Example 17—Gene Construction, Expression, and Purification ofKynureninase from Chlamydophila pecorum

A gene for expression of the kynureninase enzyme from Chlamydophilapecorum (Cp-KYNU) was synthesized using E. coli-codon optimized geneblocks. The full-length gene includes an N-terminal NcoI restrictionenzyme site (nucleotides 1-6), a start codon (nucleotides 3-5), anN-terminal His₆ tag (nucleotides 6-35), an E. coli codon optimizedCp-KYNU gene (nucleotides 36-1295), a stop codon (nucleotides1296-1298), and a C-terminal EcoRI restriction enzyme site (nucleotides1299-1304) (SEQ ID NO: 53). The aforementioned restriction enzyme siteswere used to clone the assembled gene into a pET-28a+ vector (Novagen).This construct was then used to transform BL21 (DE3) E. coli forexpression. Cells were grown at 37° C. with shaking at 210 rpm inTerrific Broth (TB) media with 50 mg/L of kanamycin. Expression wasinduced when an OD₆₀₀˜1.0 was reached by adding IPTG (0.5 mM finalconcentration) with continued shaking overnight at 16° C. Cells werethen harvested by centrifugation and re-suspended in lysis bufferconsisting of 50 mM sodium phosphate, pH 7.4, 300 mM NaCl, 0.5 mMpyridoxyl phosphate (PLP), 1 mM phenylmethylsulfonylfluoride, and 1μg/mL DNase. Lysis was achieved by French press and the lysate wascleared of particulates by centrifuging at 20,000×g for 1 h at 4° C. Thesupernatant was then filtered through a 5 μm syringe filter and appliedto a Ni-NTA/agarose column (Qiagen) pre-equilibrated in 50 mM sodiumphosphate, pH 7.4, 300 mM NaCl, and 0.1 mM PLP buffer. After loading thelysate onto the column, the resin was washed with 10 column volumes (CV)of 50 mM sodium phosphate, pH 7.4, 300 mM NaCl, and 0.1 mM PLP with 30mM imidazole. The washed enzyme was then eluted with 5 CV of PBScontaining 0.1 mM PLP and 250 mM imidazole. The eluted enzyme was bufferexchanged into fresh PBS to remove imidazole, 10% glycerol was added,and aliquots were flash frozen in liquid nitrogen for storage at −80° C.

Example 18—Kinetic Parameters of Chlamydophila pecorum Kynureninase(Cp-KYNU)

The kinetic parameters of Cp-KYNU (SEQ ID NO: 57) were quantified by aspectrophotometric assay, in which the decay in the maximum absorbanceof the enzyme substrate, L-kynurenine, was monitored as a function oftime. L-Kynurenine solutions were prepared in PBS buffer, pH 7.4, toresult in final concentrations ranging from 16 μM to 500 μM.L-Kynurenine has an extinction coefficient of 4,500 M⁻¹cm⁻¹ with aλ_(max) at 365 nm while the products of the kynureninase reaction,anthranilate and L-alanine, do not appreciably absorb at 365 nm.Reactions were initiated by adding and rapidly mixing enzyme solutions(200 nM final concentrations) with the substrate solutions andmonitoring the loss of substrate at 25° C. by measuring Abs_(365 nm)over time. The resulting data were processed and fitted to theMichaelis-Menten equation for determining kinetic constants. Cp-KYNU wasdetermined to have a k_(cat)/K_(M)=3×10⁴ M⁻¹s⁻¹.

Example 19—Pharmacological Preparation of Kynureninase fromMucilaginibacter paludis

To improve the circulation time of the enzyme in vivo, the hydrodynamicradius of Mu-KYNU was increased by functionalizing surface reactivegroups in the protein by conjugation to PEG. In one embodiment, Mu-KYNUwas PEGylated by reaction of surface lysine residues with Methoxyl PEGSuccinimidyl Carbonate 5000 MW (NANOCS). The purified Mu-KYNU, wasdetermined to contain very low endotoxin levels (<20 EU/mg) as describedbelow. It was thoroughly buffer exchanged into freshly prepared 100 mMsodium phosphate buffer, pH 8.4, and concentrated to greater than 1mg/mL. The resultant solution was added directly to a 100:1 molar excessof solid PEG reagent and allowed to react at room temperature for 1 hwith stirring. Un-reacted PEG was removed from solution by thoroughbuffer exchange into fresh, endotoxin-free PBS in a 100 kDa cutoffcentrifugal filtration device (Amicon). The apparent molecular mass ofthe enzyme was then checked on a size exclusion HPLC column (Phenomenex)in PBS using a MW standard solution from BioRad to generate a standardcurve, and enzyme retention times were compared to those of the proteinstandards. Endotoxin levels were quantified using the Chromo-LAL kineticchromogenic endotoxin testing kit (Associates of Cape Cod, Inc.).

Example 20—Enhanced L-Kynurenine Degradation in an Engineered HumanKynureninase Variant

The h-KYNU enzyme is highly selective towards the hydrolysis of 3′-OHkynurenine and has about 1,000 fold lower catalytic activity towardsL-kynurenine. Because of its poor catalytic activity towardsL-kynurenine, the wild-type human enzyme is not suitable for therapeuticpurposes. To engineer improved L-kynurenine degrading activity intoh-KYNU, a saturation mutagenesis library was constructed by overlapextension polymerase chain reaction (PCR) using the h-KYNU gene and apair of oligonucleotides designed to introduce mutations of the codoncorresponding to amino acid F306. F306 is located within the active siteof h-KYNU where it appears to play a role in substrate binding. The F306saturation library was screened for activity using the microtiter platekynureninase assay of Example 6. More than a dozen clones displayedsignificantly higher activity than wild-type h-KYNU and were selectedfor further analysis. Sequencing of these clones revealed that two aminoacid substitutions at position F306 resulted in increased L-kynureninedegrading activity, namely h-KYNU-F306M (SEQ ID NO: 55) and h-KYNU-F306L(SEQ ID NO: 56). These variants were then purified to homogeneity and adetailed kinetic analysis revealed a 2-fold and 5-fold increase ink_(cat)/K_(M) for L-kynurenine for h-KYNU-F306M and h-KYNU-F306L,respectively, as compared to wild-type h-KYNU.

Example 21—Comparison of Pf-KYNU, Anti-PD1, and Anti-CTLA-4 Therapies inthe Autologous B16 Mouse Melanoma Model

The PEGylated Pseudomonas fluorescence kynureninase (PEG-Pf-KYNU) wasevaluated in the B16 melanoma mouse model in a side-by-side comparisonwith the anti-PD1 (clone RMP1-14, BioXCell # BE0146) or anti-CTLA-4(clone UC10-4F10-11, BioXCell # BE0032) immune checkpoint inhibitorantibodies. Fifty thousand B16 cells were implanted in the flank ofC57BL/6J mice (Day 0, n=8 mice each group). Once palpable tumorsdeveloped (Day 10), the animals were treated with either 250 μganti-PD1, 100 μg anti-CTLA-4 (200 μg 1^(st) dose as per Holmgaard et al.(2013)), or 500 μg of PEG-Pf-KYNU at the times shown (FIG. 8).Heat-inactivated PEG-Pf-KYNU was used as a control. Administration ofPEG-Pf-KYNU resulted in significant tumor growth retardation andextended survival in a manner indistinguishable from that observed withthe anti-PD1 or anti-CTLA-4 checkpoint inhibitor antibodies (FIG. 8) forPEG-Pf-KYNU vs. inactivated enzyme or PBS only.

Example 22—Efficacy of Mu-KYNU or Pf-KYNU and Anti-PD1 CombinationTherapy in the Autologous B16 Mouse Melanoma Model

The PEGylated enzymes (PEG-Pf-KYNU and PEG-Mu-KYNU) were evaluated inB16 melanoma allografts in combination with the anti-PD1 immunecheckpoint inhibitor antibody (Curran et al., 2010). Four groups ofC57BL/6J mice (10 per group) were implanted with 50,000 B16 cells (Day0) and tumors were allowed to develop. Once palpable tumors developed(Day 10), the animals were treated with 250 μg anti-PD1 by IP injection(clone RMP1-14, BioXCell # BE0146) on days 10, 13, and 16 either with orwithout 500 PEG-Pf-KYNU or 500 μg PEG-Mu-KYNU s.c. near the tumor site.Mice received a total of six doses of KYNU between days 10 and 25. Onegroup was given PBS injections i.p. as a control for PD-1. Tumor growthwas drastically impaired or even reversed in all treatment arms comparedto PBS control (FIG. 9A). Importantly, additive effects were observedwith anti-PD1 in combination with KYNU resulting in complete remissionof 60% of the tumors with PEG-Pf-KYNU/anti-PD1 treatment and 20% of thetumors with PEG-Mu-KYNU/anti-PD1 treatment (FIG. 9B). CorrespondingKaplan-Meier plots are provided in FIG. 9C.

Example 23—Efficacy of PEG-Mu-KYNU Therapies in the Autologous B16 MouseMelanoma Model

The PEGylated Mucilaginibacter paludis kynureninase (PEG-Mu-KYNU) wasevaluated in the B16 melanoma mouse model. Allografts were initiated byimplanting 50,000 B16 cells in the flanks of C57BL/6J mice (Day 0, n=9mice per group). Once palpable tumors developed (Day 10), the animalswere treated with 500 μg of PEG-Mu-KYNU by subcutaneous injection nearthe tumor site every three days for a total of 6 doses. An identicaltreatment regimen with heat-inactivated PEG-Mu-KYNU was used as acontrol. Administration of PEG-Mu-KYNU resulted in tumor growthretardation (FIG. 10A) with an extended median survival time of 25 dayscompared to 22 days for the heat-inactivated PEG-Mu-KYNU control (FIG.10B).

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1.-51. (canceled)
 52. An isolated kynureninase enzyme having an aminoacid sequence that is at least 90% identical to SEQ ID NO: 8, whereinthe enzyme is formulated in a pharmaceutically acceptable carrier. 53.The enzyme of claim 52, having one or more substitutions relative to SEQID NO: 8, wherein the one or more substitutions comprise a substitutionfor the Phe found at position 306 of SEQ ID NO:
 8. 54. The enzyme ofclaim 52, wherein the enzyme has an amino acid sequence that is at least95% identical to SEQ ID NO:
 8. 55. The enzyme of claim 53, wherein theat least one substitution comprises Phe306Met.
 56. The enzyme of claim53, wherein the at least one substitution comprises Phe306Leu.
 57. Theenzyme of claim 52, further comprising a heterologous peptide segment.58. The enzyme of claim 57, wherein the heterologous peptide segment isan XTEN peptide, an IgG Fc, an albumin, or an albumin binding peptide.59. The enzyme of claim 52, wherein the enzyme is coupled topolyethylene glycol (PEG).
 60. The enzyme of claim 59, wherein theenzyme is coupled to PEG by way of one or more Lys or Cys residues. 61.The enzyme of claim 52, wherein the kynureninase has greater catalyticactivity towards kynurenine than 3′-OH kynurenine.
 62. The enzyme ofclaim 52, wherein the kynureninase has a k_(cat)/K_(M) for kynurenine ofat least 0.5 M⁻¹/s⁻¹.
 63. A nucleic acid encoding the enzyme of claim52.
 64. The nucleic acid of claim 63, wherein the nucleic acid is codonoptimized for expression in bacteria, fungus, insects, or mammals. 65.An expression vector comprising the nucleic acid of claim
 63. 66. A hostcell comprising the nucleic acid of claim
 63. 67. The host cell of claim66, wherein the host cell is a bacterial cell, a fungal cell, an insectcell, or a mammalian cell.
 68. A pharmaceutical composition comprisingthe expression vector of claim
 65. 69. The pharmaceutical composition ofclaim 68, wherein the enzyme has an amino acid sequence that is at least95% identical to SEQ ID NO:
 8. 70. The pharmaceutical composition ofclaim 68, wherein the kynureninase has greater catalytic activitytowards kynurenine than 3′-OH kynurenine.
 71. The pharmaceuticalcomposition of claim 68, wherein the kynureninase has a k_(cat)/K_(M)for kynurenine of at least 0.5 M⁻¹/s⁻¹.